WO1994019463A2 - Developmental tyrosine kinases and their ligands - Google Patents

Abstract

The invention relates to mammalian receptor tyrosine kinases designated developmental tyrosine kinases (Dtks). Dtks are expressed in multipotential haematopoietic cells, in embryonic stem cells, in brain tissue and in testis, but are not expressed mature lineage-restricted haematopoietic cells. The invention provides full-sequence Dtks as well as extracellular receptor domains of such Dtks. The invention further provides nucleic acid molecules encoding such Dtks, vectors containing DNA encoding such Dtks, ligands which bind to such Dtks, and methods of therapeutic and/or prophylactic treatment employing either the ligands or extracellular receptor domains.

Description

DENELOPMEΝTAL TYROSINE KINASES AND THEIR LIGANDS

FIELD OF THE INVENTION

The present invention generally relates to protein tyrosine kinase receptors widely expressed by early cells of the haematopoietic system, by cells of the neuronal system in brain tissue, and in testis, ligands for such receptors and nucleic acid molecules encoding such receptors.

BACKGROUND OF THE INVENTION There are several parallels between the development of the haematopoietic and neuronal systems. In particular, the presence of regulatory protein molecules termed growth factors which recognise and bind to specific cell membrane receptors is a common feature of these two systems. It is possible that shared families of receptors exist that are expressed in both early haematopoietic and neuronal stem cells. In turn, there may be a family of proteins which bind these receptors and function as stem cell growth factors.

The current view of vertebrate haematopoietic ontogeny holds that a succession of pluripotential stem cell migrations originate in the yolk sac blood islands, initially invade the hepatic rudiment, and then the spleen and bone marrow. From the bone marrow, a limited number of multipotential stem cells are laid down during embryogenesis that give rise to a much larger population of developmentally restricted progenitor cells, and ultimately produce the mature cells of at least eight cell lineages. The cells of these lineages are classified as red and white blood cells. The white blood cells contain the mature cells of the lymphoid and myeloid systems. Lymphoid cells contain T and B lymphocytes and are derived from pre-T and pre-B cells, respectively. The myeloid system comprises several cell types known as granulocytes, platelets, monocytes, macrophages, and megakaryocytes. The granulocytes are further divided into neutrophils, eosinophils, basophils and mast cells (see review by Metcalf D. The Molecular Control of Blood Cells.

Harvard Univ Press, 1988). The haematopoietic system functions by precisely controlling the production of cells in the various lineages. Totipotent haematopoietic stem cells have the ability to both self-renew and differentiate. Stem cells undergo a series of differentiation steps leading to increasingly lineage-restricted progenitor cells. The more mature progenitor cells are restricted to production of only one or two lineages. For some time the colony-forming unit-spleen (CFU-S) assay served to operationally define all stem cells. Recent evidence demonstrates heterogeneity within CFU-S, with only a small fraction of CFU-S capable of contributing to long-term repopulation following ablation of the haematopoietic system by irradiation. It is recognised that stem and progenitor populations are not discrete, but represent a continuum of cells from those of high self-renewal capacity and low probability of differentiation to those cells with low self-renewal probability and high commitment to differentiation. When long-term haematopoiesis is investigated at the clonal level, studies have shown that single stem cell clones are sufficient to maintain haematopoiesis over the lifetime of an animal.

The development of the mammalian embryo is governed by interactions between different embryonic cell populations. This process is manifest at the cellular level in the precise temporal and spatial control of proliferation, differentiation and migration. The coordination of these processes may be achieved in part by the action of a family of regulatory molecules termed growth factors. Growth factors can evoke diverse responses in different cell types and may interact with one another synergistically or antagonistically. Their action is complex and most of our current understanding results from in vitro experiments. In most instances, haematopoietic growth factor actions defined in vitro have been confirmed in vivo.

In haematopoiesis, some growth factors are lineage-restricted in their action. These include erythropoietin that acts predominantly on red cell development, and granulocyte colony-stimulating factor that^* s predominant action is on granulocytes. At the other end of the spectrum is interleukin-3 which can act on several target cells such as granulocyte-macrophage progenitors, eosinophils, megakaryocytes, erythroid cells and mast cells. There are no known growth factors that function exclusively on haematopoietic stem cells. The ligand for c-kit, termed stem cell factor, kit ligand or mast cell growth factor is the product of the Steel (SI) locus in mice. The factor acts either alone or synergistically with several known growth factors on primitive stem cells. It is believed that this factor is essential for the development of early haematopoietic stem cells, and cells of the erythroid and mast cell lineages.

The stem cell compartment may be viewed as a finely tuned balance between the action of inhibitors and the stimulatory role of cytokines. As with other stem cell systems, haematopoietic stem cells are distributed in a defined spatial manner within adult bones and not in a random, homogeneous mixture of interacting cell types. A concept that underlies the regulation of haematopoietic stem cell development is that these cells reside within a specialised microenvironment, where the regulatory signals act locally. Stromal cells constitute the bone marrow microenvironment.

Embryonic stem cells are permanent cell lines established directly from the inner cell mass of the preimplantation mouse embryo. They retain the ability to participate in normal embryonic development and, following introduction into the blastocyst, generate chimaeric animals that are mosaic in all tissues. Embryonic stem cells are increasingly being used as cellular vectors for experimentally manipulating the mouse genome.

Doetschman has demonstrated that embryonic stem cells can generate primitive erythroid cells in culture (Doetschman et. al. J. Embryol. Exp. Morphol. 87, 27-45, (1985)). This result was achieved by inducing embryonic stem cells to form cystic embryoid bodies in the presence of preselected batches of human cord serum.

In addition to haematopoietic cell development, it has been noted that neurons also arise in differentiating embryonic stem cells. Haematopoietic differentiation in this system occurred infrequently, slowly and was not synchronized. Recently a modified system enabling the differentiation of embryonic stem cells in methylcellulose into multiple haematopoietic lineages has been described by Wiles and Keller (Development 111. 259-267, (1991)). Using this approach, macrophages, neutrophils, erythroid cells and mast cells develop in a synchronous manner with high frequency in the absence of human cord serum. The development of haematopoiesis from embryonic stem cells in methylcellulose cultures parallels the onset of haematopoiesis in the developing mouse embryo.

.An important objective in the field of developmental biology is the identification of genes, the products of which mediate regulatory signals required during embryogenesis. There is compelling evidence that genes encoding receptor tyrosine kinases (RTKs) are involved in early development in vertebrates. The general family of protein tyrosine kinases can be recognised by the presence of several conserved amino acid regions in the catalytic domain. These conserved regions have been summarised by Hanks et al (Science 241. 42-52, (1988)) and by Wilks et al (Proc. Νatl. Acad. Sci. USA 86. 1603-1607, (1989)). The receptor for macrophage colony-stimulating factor c-frns, which is important in myeloid cell differentiation and placental development is an RTK. The mouse developmental mutation W has been shown to involve an RTK. The W locus encodes the c-kit RTK and affects the proliferative and/or migratory properties of primordial germ cells, melanoblasts and haematopoietic stem cells. A recently described RTK termed flk-2, which is related to c-kit, has been isolated using the polymerase chain reaction (PCR) with oligonucleotides to conserved kinase domain motifs. Messenger RΝA transcripts for flk-2 are expressed in populations enriched for stem cells and primitive uncommitted progenitor cells, and are absent in mature haematopoietic cells (see Matthews et al. Cell 65. 1143-1152, (1991)).

Additional receptor tyrosine kinases expressed on pluripotential haematopoietic stem cells are needed to facilitate the in vitro growth of stem cells. The nucleic acid molecules that encode receptor tyrosine kinases expressed by pluripotential stem cells are needed to produce recombinant receptors and ligands.

In vertebrate development, the cells whose descendants give rise to the nervous system are first identified as the neural ectoderm. This forms a tube-like structure beneath the surface of the ectoderm. Following closure of the neural tube some precursor cells detach from the apical neural tube and form a transient structure called the neural crest. These cells rapidly disperse into the embryo along complex migratory pathways. The proliferating neural crest cells also invade developing tissues such as the skin, gut, and the adrenal gland to form differentiated cell populations within these tissues; eg. melanocytes, enteric neurons and adrenal medullary chromaffin cells.

The diversity of cell types derived from the neural crest poses the problem of how uncommitted embryonic cells acquire particular developmental fates. There are strong parallels between neural crest cell lineage diversification and the process of haematopoiesis. It has been proposed that the earliest neural crest cells should be multipotent and maybe capable of self renewal. Secondly, it should be possible to identify committed progenitors that proliferate symmetrically and are restricted to distinct sublineages and thirdly, there should exist factors which influence the proliferation and/or differentiation of specific types of progenitors (see Anderson

Neuron 3. 1-12, (1989)).

Soluble proteins variously termed neurotrophic, growth, and neuronal differentiation factors have been identified that influence the developmental growth, maintenance of function, and plasticity of neuronal populations. These factors have been implicated in the proliferation and differentiation of neurons during embryonic development and in their growth and survival in the adult nervous system. There are a growing number of neurotrophic factors, including nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4. These molecules constitute a closely related family sharing at least 60% amino acid identity. If the parallel to the haematopoietic system is extended, the range and complexity of cells derived from the neural crest implies that there will be a large number of protein regulators which control this system.

Two different types of receptors have been demonstrated for neurotrophins. One group of these receptors are transmembrane glycoproteins with tyrosine kinase activity encoded by members of the trk protooncogene family. It would therefore be important to isolate additional receptor tyrosine kinases from developing systems such as embryonic stem cells which contain neurons. Ligands for such receptors are required to act inter alia as neurotrophic factors. Nucleic acid molecules encoding the receptors and ligands are needed to produce recombinant receptors and ligands.

It is the object of the present invention to go some way towards fulfilling the above objectives or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION

The present invention has a number of aspects. In a first aspect, the invention provides a mammalian receptor tyrosine kinase which is a developmental tyrosine kinase (Dtk) and which is expressed in multipotential haematopoietic cells, in embryonic stem cells, in brain tissue and in testis, but which is not expressed in mature lineage-restricted haematopoietic cells.

In a further aspect, the invention provides an extracellular receptor domain of a receptor tyrosine kinase as defined above. In preferred embodiments, this extracellular receptor domain can be bound to a support, or can be in a soluble form.

In still a further aspect, the invention provides a nucleic acid molecule encoding a receptor tyrosine kinase or extracellular receptor domain as defined above. This nucleic acid molecule is preferably DNA.

In yet a further aspect, the invention provides a vector including a DNA molecule as defined above.

In still a further aspect, the invention provides a method of producing a receptor tyrosine kinase comprising the steps of:

(a) culturing a host cell which has been transformed or transfected with a vector as defined above to express the encoded receptor tyrosine kinase or extracellular receptor domain; and (b) recovering the expressed receptor tyrosine kinase.

As yet an additional aspect, the invention provides a ligand that binds to a receptor tyrosine kinase as defined above.

The ligand can take two forms. In one form, the ligand stimulates the proliferation, differentiation and/or survival of cells which express a receptor tyrosine kinase as defined above (a stimulant ligand).

In the second form, the ligand is antagonistic and at least partially blocks or inhibits the function of a receptor tyrosine kinase as defined above through binding to said receptor (an antagonistic ligand).

In another aspect, the invention provides a method of stimulating the proliferation, differentiation and/or survival of a cell expressing a receptor tyrosine kinase as defined above comprising contacting the cell with a stimulant ligand as defined above.

In yet a further aspect, the invention provides a method of inhibiting the function of a receptor tyrosine kinase as defined above comprising contacting the receptor with an antagonistic ligand as defined above.

In still another aspect, the invention provides a method of treating a disease, syndrome or condition caused or mediated by an excess of a ligand as defined above comprising the step of contacting said excess of said ligand with an effective amount of a receptor tyrosine kinase or an extracellular receptor domain as defined above.

In another aspect, the invention provides a method of extracting a ligand from a medium which may contain said ligand comprising the step of contacting said medium with a receptor tyrosine kinase or with an extracellular receptor domain as defined above. The invention also provides a method of isolating ligand(s) from a medium which may contain said ligand(s), comprising the steps of:

(a) contacting said medium with an effective amount of a receptor tyrosine kinase or an extracellular domain as defined above; (b) detecting which ligand(s) bind; and

(c) isolating such bound ligand(s).

While the invention broadly consists in the foregoing, it should be appreciated that it also includes the more specific embodiments detailed in the following description:

DESCRIPTION OF THE FIGURES

Figure 1 shows expression of murine Dtk in embryonic stem (ES) cells and embryoid bodies. RNase protection analysis was performed on total RNA (10 μg) from ESD3 ES cells growing in Leukaemia Inhibitory Factor (LIF) (day 0), or from ES cells maintained in the absence of LIF that were differentiating and developing into cystic embryoid bodies (days 2 to 18). As a control tRNA (10 μg) was also used. The markers were pBR322 digested with Msp I. The size of the free murine Dtk probe was 229 nt. A fully protected fragment representing the presence of murine Dtk transcripts was 187 nt in length. The free β-actin protected fragment is shown in each lane as an RNA loading control.

Figure 2 shows expression of murine Dtk in embryonic mouse tissues. RNase protection analysis was performed on total RNA (10 μg) isolated from E14.5 embryonic tissues of the C57BL/6J mouse strain. Details of the markers, probes and controls are as described for Figure 1.

Figures 3 and 4 show expression of murine Dtk in adult mouse tissues. RNase protection analysis was performed on total RNA (10 μg) isolated from the various tissues of adult C57BL/6J mice. Details of the markers, probes and controls are as described for Figure 1. Figure 5 shows expression of murine Dtk in murine cell lines. The most abundant expression is in the multipotential cell lines FDC-P1 and DA2, and the mast cell line P815. The majority of other cell lines are lineage-committed, mature haematopoietic cell lines, which have very limited murine Dtk expression. The ΝIH 3T3 cell line is derived from embryonic fibrobl-asts and C2C12 is a myoblast cell line.

Figure 6 shows the cDΝA and amino acid sequence of murine Dtk.

Figure 7 shows the cDΝA and amino acid sequence of human Dtk.

DETAILED DESCRIPTION OF THE INVENTION A. Receptors

In a first aspect, this invention provides a mammalian receptor protein tyrosine kinase (PTK). The mammal in which the PTK exists may be any mammal, such as a mouse, rat, rabbit or human.

Members of the PTK family are recognised by the conserved amino acid regions in the catalytic domains. Examples of PTK consensus sequences have been provided by Hanks et al. (Science 241 42-52 (1988), especially Figure 1 starting at page 46) and by Wilks et al (Proc. Natl. Acad. Sci. USA 86. 1603-1607 (1989), especially Figure 2 on page 1605).

Hanks et al. identify eleven catalytic subdomains containing PTK consensus residues and sequences. The PTKs of the present invention contain most or all of these consensus residues and sequences.

As indicated above, the PTKs of the invention are receptor PTKs and so are also generally referred to as RTKs. Further, as the applicants believe that the RTKs of the invention are involved in mammalian cell development, they are specifically referred to hereinafter as developmental tyrosine kinases (Dtks). The Dtks of the invention are transmembrane receptor tyrosine kinases whose extracellular domains contain two immunoglobulin-like motifs followed by two fibronectin-type III repeats. RTKs of this structure (Axl(Ufo,Ark)) are already known (Janssen et al, Oncogene 6, 2113-2120 (1991); O'Biyan et al, Mol. Cell. Biol 11. 5016-5031 (1991); Rescigno et al Oncogene 6. 1909-1913 (1991); Faust et al- Oncogene 7. 1287-1293 (1992)). The Dtks of the invention are however distinguished from those RTKs having the equivalently structured extracellular domains by their potential function based upon their distribution within the mammalian body.

With regard to this latter feature of the Dtks of the invention, the applicants have conducted experiments to determine the range of cells in which the developmental tyrosine kinases of the invention are expressed. These experiments were specifically performed in relation to murine Dtk but are believed to be illustrative of the expression of all mammalian Dtks of the invention.

A.1 Analysis of Murine Dtk expression

The expression of murine Dtk in a range of embryonic and adult mouse tissues was analyzed by ribonuclease protection analysis, using a probe that encompassed sequences encoding the membrane-proximal portion of the extracellular domain of the receptor.

Materials and Methods

1. Embryonic stem cell culture The ESD3 embryonic cell line (Doestschman et al., J. Embryol. Exp. Morphol._.87

27-45 (1985)) was maintained on gelatin-coated dishes in Dulbecco's-modified Eagle's medium (DMEM) with additives according to established procedures (Hogan et al., Cold Spring Harbour Laboratory, 1-332 (1986)), in the presence of LIF. Cystic embryoid bodies were established following collagenase treatment of the ES cells and subsequent suspension culture in bacteriological-grade petri dishes in DMEM with additives in the absence of LIF (Wiles and Keller, Development 111. 259-265 (1991)). 2. Fetal liver haematopoietic stem cell enrichment

Low density haematopoietic stem cells were isolated from an E14.5 fetal liver cell suspension using equilibrium density centrifugation on a discontinuous metrizamide gradient according to the method of Visser et al., J. Exp. Med..59, 1576-1590 (1984). Following this procedure, low density fetal liver cells (p24

< 1.078 g/cm³) were incubated for 20 minutes on ice in DMEM medium with 5 μg/10⁶ cells of AA4 monoclonal antibody (rat IgG_2b; McKearn et al., Proc. Natl. Acad. Sci. USA. 82, 7414-7418 (1985)) and washed twice. This antibody has been shown to recognise the most primitive haematopoietic stem cell in fetal liver (Jordan et al., CeU, 61, 953-963 (1990)). The AA4 labelled cells were then incubated on ice for 20 minutes with magnetic beads conjugated with anti-rat IgG antibody as outlined in the manufacturer's protocol (Advanced Magnetics Corp., Cambridge, MA). Following incubation, AA4⁺ cells were positively-selected on a magnet. Stem cell enrichment was assessed by re-labelling the cells with the AA4 antibody, followed by a second layer antibody staining with goat anti-rat fluorescein isothiocyanate and flow cytometric analysis on a FACS 440 (Becton Dickinson, San Jose, CA).

3. RNA analysis RNase protection analysis was performed by hybridization of 10 μg of total RNA to RNA probes that encoded sequences of murine Dtk and β-actin, overnight at 52°C. RNase digestion was performed with RNase Tl (1.75 g/ml) and RNase A (35 μg/ml) at 37°C for one hour. The reaction was stopped with proteinase K (333 / g/ml) and SDS (0.3%). The products were run on a 6% urea/acrylamide gel and the autoradiograph exposed at -70°C. The probe for analysis of Dtk expression was derived from nucleotides 1158 to 1334 of the Dtk sequence, a segment which encodes the membrane-proximal portion of the extracellular domain, and which had been subcloned into pGEM-4Z. In an RNase protection assay, the free probe yielded a 229 nucleotide (nt) band, and Dtk transcripts protected a fragment of 187 nt. A riboprobe was also constructed from a Sal

I-Sma I fragment of human β-actin. The length of the free β-actin probe was 132 nt and β-actin transcripts protected a 54 nt fragment. Results

1. Embryonic stem cells

Figure 1 demonstrates the expression of Dtk transcripts in both totipotent ES cells growing in LIF (termed day 0), and in differentiating cystic embryoid bodies growing in the absence of LIF for up to 18 days. In this developmental system

Dtk is expressed almost uniformly from days 0 to 18, indicated by the presence of a protected 187 nt band for each RΝA analyzed. The two bands of approximately 220 nt and 210 nt present in lanes for each RΝA sample analyzed are also present in the tRΝA lane and are regarded as nonspecific. Of considerable interest with regard to the importance of this receptor in mouse development is the demonstration of Dtk expression in totipotent ES cells. The ES cells from which RΝA was extracted for day 0 analysis were selected from cultures, following morphological assessment by phase-contrast microscopy to confirm that they were undifferentiated.

2. Embryonic tissues

Expression of Dtk was detected in total RΝA isolated from a wide range of mid- gestational E14.5 embryonic mouse tissues including the brain, eye, thymus, lung, intestine, forelimb, hindlimb and testis (Figure 2). There was limited expression in heart and unfractionated liver.

Figure 2 shows enrichment of Dtk transcripts in E14.5 fetal liver low density AA4⁺ haematopoietic stem cells. Following density-gradient centrifugation and positive selection, the cells used for RΝA analysis were greater than 95% AA4⁺, as assessed by flow cytometry (data not shown). Dtk expression was also detected in day 14.5 placenta.

3. Adult tissues

In contrast to the widespread expression of Dtk in embryonic tissues, the pattern of expression in adult tissues becomes restricted (Figures 3 and 4). Dtk transcripts were most abundant in brain, esophagus, bladder, testis, and ovary. In brain, expression of Dtk (relative to β-actin) was more abundant in adult than in embryonic tissue. Adult tissues which contained less abundant, but detectable transcripts were lung, and regions of the gastrointestinal tract including the stomach and both the small and large intestine. Tissues in which Dtk transcripts were undetectable 01 expressed at extremely low levels included the salivary gland, thymus, heart, liver, skeletal muscle, kidney, spleen, bone marrow, adrenal gland and uterus.

4. Murine cell lines

The pattern of expression of Dtk in murine cell lines was analyzed in relation to the following: WEHI-3B, 416B, EL4, S03, SP2/0, P388D-,, P815, FDC-P1, DA2, FDC-P1/IL-2 ras, NIH3T3 and C2C12. The results are shown in Figure 5.

As can be seen from Figure 5, the results are consistent with those above, with the most abundant expression being in the multipotential cell lines FDC-P1 and DA2, and in mast cell line P815. Significant expression is also observed in myoblast cell line C2C12.

In contrast, the remaining cell lines (lineage-restricted mature haematopoietic cell lines) show very limited murine Dtk expression.

From this analysis, the applicants have derived the condition defining the Dtks of the invention - they are expressed in multipotential haematopoietic cells, in totipotent embryonic stem cells, in brain tissue and in testis, but not in mature lineage-restricted haematopoietic cells.

For the purpose of this specification, a multipotential haematopoietic cell is an early haematopoietic cell. Examples of multipotential haematopoietic cells include multipotential factor-dependent cells that have the capacity to proliferate and differentiate into mature haematopoietic cells. In contrast, a mature haematopoietic cell is non self-renewing and has limited ability to give rise to multiple cell lineages. Mature lineage-restricted haematopoietic cells, for the purposes of this specification, are therefore represented by haematopoietic cell lines of the T or B lymphoid lineage or mature myeloid lineages. The Dtks of the present invention may or may not be expressed in intermediate cells poised between the state of being multipotential and mature.

In terms of brain tissue, the Dtks of the invention are primarily expressed in neuronal cells.

In terms of testis, the Dtks are primarily expressed in the Sertoli cells.

It will of course be appreciated by those persons skilled in this art that the reference to the Dtks of the invention not being expressed in mature lineage- restricted haematopoietic cells is in a biological context and does not mean that there is absolutely no expression of the Dtk in these cells. As is apparent from Figures 1 to 5, what is meant by the phrase "not expressed in mature-lineage restricted haematopoietic cells" is that there is no significant expression of the Dtk in the cell, i.e. that expression is either undetectable or at an extremely low level.

The restricted expression of the Dtks of the invention to cells representative of early multipotential cells, with substantial absence of expression in lineage- restricted cells such as T or B lymphocytes, is consistent with this receptor functioning and transducing signals from the microenvironment to the haematopoietic stem cell compartment. The expression of the Dtk in embryonic stem cells and in some fetal tissues such as brain is also consistent with this receptor and its ligand having a functional role in the specification of cell lineages during embryonic development, including neuronal development. Furthermore, the receptor and its ligand is likely to have a role in the maintenance of function and plasticity in neuronal populations or their derivatives. Finally, the expression of the receptor in adult brain is consistent with the receptor and its ligand having a role in the growth and survival of neurons in the adult nervous system.

The embryonic stem cell and haematopoietic multipotential cell line mRNA for

Dtk migrates relative to 28S and 18S ribosomal bands on formaldehyde agarose gels at approximately 4.2 Kb. In adult brain tissues, Dtk mRNA migrates at approximately 4.2 Kb. The Dtks of the invention can usefully be provided in a number of different forms. These include the Dtk itself, the "mature" form of the Dtk, and the extracellular receptor domain of the Dtk.

The "mature" form of the Dtk of the invention is the Dtk less its native amino- terminus leader or signal sequence, whereas the extracellular receptor domain is the Dtk lacking the transmembrane region and catalytic domain.

The extracellular domain may be identified through commonly recognised criteria of extracellular amino acid sequences. The determination of appropriate criteria is known to those skilled in the art, and has been described, for example by Hopp et al.. Proc. Νatl. Acad. Sci. USA 78. 3824-3828 (1991); Kyte et al., J. Mol. Biol. 157. 105-132 (1982); Emini. J. Virol 55. 836-839 (1985); Jameson et al. CA BIOS 4, 181-186 (1988); and Karplus et al. Νaturwissenschaften 72. 212-213 (1985). Amino acid domains predicted by these criteria to be surface exposed are characteristic of extracellular domains.

The Dtks of the invention or their extracellular receptor domains may be prepared by methods known in the art. Such methods include protein synthesis from individual amino acids as described by Stuart and Young in "Solid Phase Peptide

Synthesis", Second Edition, Pierce Chemical Company (1984). It is however preferred that the Dtks and/or their extracellular receptor domains be prepared by recombinant methods as will be detailed hereinafter.

A.2 Specific Dtks of the Invention

A.2.1 Murine Dtk

As is indicated above, a first Dtk of the invention, murine Dtk, has been identified in certain tissues of the mouse. Murine Dtk generally has the nucleic acid and deduced amino acid sequence shown in Figure 6. Figure 6 represents individual amino acid residues as single letters as follows: Three-letter One-letter

Amino Acid abbreviation symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn Ν

Aspartic acid Asp D

Asparagine or aspartic acid Asx B

Cysteine Cys C

Glutamine Gin Q

Glutamic Acid Glu E

Glutamine or glutamic acid Glx Z

Glycine Gly G

Histidine His H

Isoleucine He I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan T W

Tyrosine Tyr Y

Valine Val V

Details of the sequence of murine Dtk are as follows.

Sequence Analysis of the Murine Dtk

Figure 6 shows the 3.919 Kb nucleotide and deduced amino acid sequence for murine Dtk from murine neonatal brain. Within the 5' region, a potential site for translation initiation (-GGAGCATGGGG-) is found within a good Kozak consensus sequence. The first methionine imtiates an open reading frame of 874 amino acids. Using the method of von Heijne Sequence Analysis in Molecular Biology 113-117, San Diego, Academic Press (1987), the signal cleavage site is predicted to be between alanine 24 and alanine 25, which specifies a 24 amino acid hydrophobic leader sequence and a mature receptor tyrosine kinase protein of 850 amino acids. Amino acids AGLK to PHSR form a 386 amino acid extracellular domain. A 25 amino acid hydrophobic region from TSWV to LILL is consistent with that of a transmembrane domain (Fasman and Gilbert, Trends Biochem 15. 89-92 (1990)), while the remaining amino acids ending HSSC comprise the cytoplasmic domain.

The extracellular domain of murine Dtk contains eight consensus sites (NxT or S) for N-Iinked glycosylation, predicting that the mature Dtk protein is glycosylated. Within the extracellular domain, two repeating protein motifs are identifiable.

Using the predictive methods of Williams and Barclay, Ann. Rev. Immunol 6, 381-405 (1988), two C-type immunoglobulin (Ig)-like domains are present from amino acids KLMG to GEET (Ig-like domain I) and FFTV to NIKG (Ig-like domain II). The first Ig domain has a structure similar to a Cl domain, while the second Ig domain is more C2-like. Based on the analysis of Petersen et al, Proc.

Natl. Acad. Sci. USA 80. 137-141 (1983), there are two fibronectin type III modules present from amino acids PPAA to PYGD (domain I) and from amino acids PFQT to SHDH (domain II).

Analysis of the 439 amino acid cytoplasmic domain sequence of murine Dtk shows many of the motifs which are highly conserved within the catalytic kinase domain of protein tyrosine kinases (Hanks et al. Science 241. 42-52 (1988)). The motifs GKGEFG and VAVK, which function as the Mg²* -ATP binding site (Ullrich and Schlessinger, CeH 61, 203-212 (1990); Cantley et al., CeH 64, 281-302 (1991)), are observed at the start of the kinase domain. Further towards the carboxy-terminus of Dtk other conserved kinase motifs are identifiable, including the motif

IHRDLAARΝ, the DFG triplet motif and the motifs KWLALES and DVWAFG.

Alignment of the kinase domain of Dtk with other protein tyrosine kinase domains including that of Ufo, suggests there is a kinase insert region specified by the amino acids RIGEΝPFΝ. There are 12 tyrosine residues within the cytoplasmic domain of Dtk, including two residues located near the C-terminus that are nested within sequences that exhibit strong homology to Src homology 2 (SH2) domain

binding sites (Songyang et al., Cell 72, 767-778 (1993)). One of these sequences, EEVYDLM, is a putative binding site for phosphatidylinositol 3-kinase, but lies within the catalytic domain proper and is unlikely to be autophosphorylated. The sequence DPLYIΝI fulfills criteria for either a Sem5/Grb2 binding site or a phospholipase C-γ binding site (Songyang et al, (1993)) supra, and its position in the C-terminal tail makes it a good candidate for phosphorylation.

In specific aspects, the invention provides murine Dtk, mature murine Dtk and the extracellular receptor domain of murine Dtk.

Murine Dtk has the amino acid sequence given as SEQ ID NO 1.

Mature murine Dtk has the amino acid sequence given as SEQ ID NO 2.

The extracellular receptor domain of murine Dtk has the amino acid sequence given as SEQ ID NO 5. The invention also includes functional equivalents of murine Dtk, mature murine Dtk and the extracellular receptor domain of murine Dtk as is described hereinafter.

A.2.2 Human Dtk

A second Dtk of the invention has been identified from human tissue. This second receptor is the human homologue of murine Dtk having all of the

structural features of murine Dtk.

The nucleic acid and deduced amino acid sequence for this receptor tyrosine kinase, hereinafter called "human Dtk", is shown in Figure 7. Sequence details are as follows.

Sequence Analysis of the Human Dtk Figure 7 shows the 4.364 Kb nucleotide and deduced amino acid sequence for the human Dtk from human fetal brain. The structural features of human Dtk closely parallel those described for murine Dtk. The signal peptide encompasses amino acids MGRP to ESAA. The mature protein extends from residues AGLK to HSSC. Within the mature protein the extracellular domain is defined by residues AGLK to PHSR, the transmembrane domain by residues TSWV to LILL, and the cytoplasmic domain from residues RKRR to HSSC.

The extracellular domain contains two repeating protein motifs made up of two immunoglobulin domains (KLMG to GGET and FFTN to ΝLKG), followed by two fibronectin type III modules (LPAA to PYAD and PFQT to SHDR). The protein tyrosine kinase domain is encompassed by the amino acids LGKG to RMEL within the cytoplasmic domain. The motifs defined within the murine protein tyrosine kinase domain are also identifiable within the human protein

tyrosine kinase domain.

Once again, in its specific aspects the invention provides different forms of the Dtk (human Dtk, "mature" human Dtk and the extracellular receptor domain of human Dtk).

Human Dtk has the amino acid sequence given as SEQ ID NO 3.

Mature human Dtk has the amino acid sequence given as SEQ ID NO 4.

The extracellular receptor domain of human Dtk has the amino acid sequence given as SEQ ID NO 6.

Once again, the invention further includes functional equivalents of human Dtk, mature human Dtk and of the extracellular receptor domain of human DTK.

A.2.3 Other Mammalian Dtks

In addition to the murine and human Dtks described above, the invention includes within its scope Dtks of other mammals. Such Dtks are the homologues of both murine and human Dtk and can be readily identified by those persons skilled in the art with reference to the characterising data given above for murine Dtk and human Dtk. By way of example, one method for identifying other Dtks of the invention involves the formation of a DΝA library from a suitable tissue source (such as brain) obtained from the mammal. This library can then be screened to identify DΝA coding for homologues to murine Dtk and human Dtk as will be described in more detail below.

B. Nucleic Acid Molecules Encoding the Dtks of the Invention In another aspect of this invention, the applicants provide nucleic acid molecules encoding the Dtks. These nucleic acid molecules may be DNA (isolated from nature, synthesised or cDNA) or RNA. Most often, the nucleic acid molecules will be DNA.

B.l Nucleic Acid Molecules Encoding Murine Dtk and Human Dtk As indicated above, the nucleic acid sequences for murine Dtk and human Dtk have been determined. In specific aspects, the invention therefore provides nucleic acid molecules (in the form of DNA) as follows:

1. A DNA molecule encoding murine Dtk having the nucleotide sequence given as SEQ ID NO 7.

2. A DNA molecule encoding mature murine Dtk having the nucleotide sequence given as SEQ ID NO 8.

3. A DNA molecule encoding the extracellular receptor domain of murine Dtk having the nucleotide sequence given as SEQ ID NO 11. 4. A DΝA molecule encoding human Dtk having the nucleotide sequence

given as SEQ ID NO 9.

5. A DNA molecule encoding mature human Dtk having the nucleotide sequence given as SEQ ID NO 10.

6. A DNA molecule encoding the extracellular receptor domain of human Dtk having the nucleotide sequence given as SEQ ID NO 12.

The invention also includes within its scope functional equivalents of these DNA molecules.

B.2 Nucleic Acid Molecules Encoding Dtks of other Mammals It will be appreciated that DNA molecules encoding the functional equivalent homologues of murine Dtk and human Dtk from other mammals are also within the scope of the invention. Such DNA molecules can be readily identified using conventional techniques and with reference to the information contained herein characterising murine Dtk and human Dtk.

By way of generic illustration, DNA molecules encoding homologues of murine

Dtk and human Dtk in other mammals can be identified by employing the following general steps:

(a) Formation of a cDNA library:

Total mRNA from a suitable tissue source (such as brain) of the mammal is prepared by standard procedures (Ausubel et al, (Eds), "Current Protocols in Molecular Biology" Greene Associates/Wiley Interscience, New York (1990)), and cDNA synthesised. A cDNA library is formed (for example in λZAP II).

(b) Library Screening:

The cDNA library formed as above is screened for the presence of cDNA encoding homologues to murine Dtk and human Dtk.

Screening will generally employ a DNA hybridisation or amplification step with the probes or primers being selected based

upon the already determined sequences of murine and human Dtk.

Most conveniently, the screening procedure will involve DNA amplification using the polymerase chain reaction (PCR) (Saiki et al Science 239. 487 (1988)) with the PCR primers being selected such that highly conserved regions from within the DNA sequence of murine and human Dtk will be within the amplified PCR product.

(c) DNA Isolation and Sequencing:

Clones from the cDNA library which are identified by screening step (b) as containing cDNA encoding homologues to murine and human

Dtk are selected, and the size of the cDNA insert sourced from the brain determined. Such clone(s) including a cDNA insert of the appropriate size to code for the full-length Dtk are selected and the cDNA insert isolated. Each isolated cDNA insert is then sequenced using known procedures (for example, using the standard dideoxy chain-termination method of Sanger et al., Proc. Νatl. Acad. Sci. USA 74. 5463-5467 (1977)).

B.3 Genetic Mapping of Murine Dtk and Human Dtk By way of further characterisation of both murine Dtk and human Dtk, the applicants have performed experiments to establish the chromosomes on which the genes coding for these Dtks are located. Details of these experiments are given below.

Materials and Methods

B.3.1 Fluorescent In Situ Hybridization (FISH)

A partial Sau3A genomic DΝA library in λ 2001, prepared from mouse ES cells (Boehm et al. Proc. Νatl. Acad. Sci. USA 88. 3927-3931 (1991)), was screened with the 3.525 kb cDΝA insert purified from pMo23A using methods previously described (Morris, et al., Blood 76, 1812-1818 (1991)). Of 34 positive clones, two of the most intensely hybridizing, λ Mo23A-7.1 and λ Mo23A-8.1, were selected for FISH studies. The pMo23A plasmid, and DΝA isolated from bacteriophage clones λ Mo23A-7.1 and λMo23A-8.1, were biotinylated by nick-translation using biotin-14-dATP (Bethesda Research Laboratories, Gaithersberg, MD). Karyotypically normal, 40,XY, mouse metaphase cells were prepared from ES cells in culture using standard procedures. Fluorescent in situ hybridization and detection procedures were essentially as described (Morris et al, Human Genetics 91, 31-36 (1993)), except that mouse Cot 1 DΝA (BRL, final concentration 250ng/μl) was used to suppress repetitive sequences in the two phage DΝA probes. Chromosomes were G-banded using DAPI (4',6-diamidino-2- phenylindoledihydrochoride, Sigma, St Louis, MO) as a counterstain for fluorescence analysis.

B.3.2 Single-strand conformation polymoφhism (SSCP) Primer sequences from the 3' untranslated region of the Dtk cDΝA used for genetic mapping were as follows:

DtkMapl 5' TGGATGGCAGTAAGGGAGG 3'

5' CTTAAGAGGGGCAAACCTGG 3' DtkMap2 5' GCTTAGAGGAGGTGAGCCAGA 3'

5' TGGGCAGTGCTGAGTTCC 3'

PCR was performed using standard conditions with the addition of ³²P-labelled dCTP. Specifically, 25 μl reactions were performed in 10 mM-Tris-HCl, 50 mM KC1 using 250 ng of genomic DΝA, 1 μM of each primer, and 1.4 mM MgCl₂.

This was overlaid with oil, denatured at 94°C for 5 minutes, and transferred to an 80°C heating block. dΝTPs were added to a final concentration of 0.2 mM, including 1.25 μCi of [α -³²P]dCTP (lμl of a 3000 Ci/mmole stock to 8 reactions). 1.25 units of AmpliTaq DΝA polymerase (Perkin-Elmer Cetus) was added and cycling conditions were as follows: 58°C annealing reaction for 1 minute, 72°C extension reaction for 2 minutes, and 91°C denaturation for 1 minute. The cycle was repeated 30 times with a final 72°C extension reaction for 5 minutes. SSCP analysis was performed by electrophoresing the single-stranded PCR products on a non-denaturing gel as follows: 2 μ\ of the PCR reaction was added to 8 μl of USB stop solution (100% formamide containing xylene cyanol and bromophenol blue). This was denatured for 5 minutes at 94°C and transferred to an ice bucket. 3μl was loaded on a 5% non-denaturing acrylamide gel containing 0.5X TBE and no

glycerol. This was run in a 4°C cold room in 0.5X TBE at 40 watts constant

power for 2-3 hours. The gel was transferred to filter paper, dried, and autoradiographed overnight with an intensifying screen.

Results

The chromosomal localisation of the gene encoding murine Dtk has been established on chromosome 2 band F using fluorescent in situ hybridisation. This result has been confirmed using single strand conformation polymoφhism analysis in the BXD recombinant inbred series.

The gene encoding human Dtk has been mapped using fluorescent in situ hybridisation to chromosome 15ql5.

C. Recombinant Expression of Dtks of the Invention

In yet another aspect, the present invention relates to the recombinant expression of the Dtks or of their extracellular receptor domains.

As will be exemplified below, the nucleic acid molecules that encode the receptors or the extracellular receptor domains of the invention may be inserted into known vectors for use in standard recombinant DΝA techniques. Standard recombinant DΝA techniques are those such as are described in Sambrook et al.; "Molecular Cloning" 2nd Edition Cold Spring Harbour .Laboratory Press (1987) and by Ausubel et al., Eds, "Current Protocols in Molecular Biology" Greene Publishing Associates and Wiley-Interscience, New York (1987).

Vectors for expressing proteins in bacteria, especially E. coli. are known. Such vectors include the PATH vectors described by Dieckmann and Tzagoloff in J.

Biol. Chem. 260. 1513-1520 (1985). These vectors contain DNA sequences that encode anthranilate synthetase (TφE) followed by a polylinker at the carboxy terminus. Other expression vector systems are based on beta-galactosidase

(pGEX); lambda P maltose binding protein (pMAL); and gluthathione S-

transferase (pGST) - see Gene 67, 31 (1988) and Peptide Research 3, 167 (1990).

Vectors useful in yeast are available and well known. A suitable example is the 2μ plasmid.

Suitable vectors for use in mammalian cells are also l nown. Such vectors include

well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and vectors derived from combination of plasmids and phage DNA

Further eucaryotic expression vectors are known in the art (e.g. PJ. Southern and P. Berg, J. Mol. Appl. Genet. I, 327-341 (1982); S. Subramani et al, Mol. Cell.

Biol. 1. 854-864 (1981); R.J. Kaufmann and P.A. Shaφ, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene." J. Mol. Biol. 159. 601-621 (1982); R.J. Kaufmann and P. A. Shaφ. Mol. Cell. Biol. 159. 601-664 (1982); S.I. Scahill et al, "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells." Proc. Νatl. Acad. Sci. USA 80. 4654-4659 (1983); G. Urlaub and L.A. Chasin. Proc. Νatl. Acad. Sci. USA 77. 4216-4220,

(1980).

The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DΝA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DΝA sequence. Examples of useful expression control sequences are the Jac system, the _.trp system, the lac system, the re system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g. the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g. Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g. the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic and eucaryotic cells and their viruses or combinations thereof.

Vectors containing the receptor-encoding DΝA and control signals are inserted into a host cell for expression of the receptor. Some useful expression host cells include well-known prokaryotic and eucaryotic cells. Some suitable prokaryotic hosts include, for example, E. coli. such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHT, and E. coli MR01, Pseudomonas. Bacillus, such as Bacillus subtilis. and Streptomyces. Suitable eucaryotic cells include yeast and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture. A specific although non-limiting example of this aspect of the invention is set out below. It will be appreciated that while the expression of murine Dtk is exemplified, the procedures disclosed are equally applicable to the expression of other Dtks, or to the expression of extracellular receptor domains of such Dtks.

C.l Expression of cloned murine Dtk in heterologous cell lines The coding region of murine Dtk was ligated in-frame into the commercially available expression vector pcDNA3 (InVitrogen) using standard molecular biology techniques. The pcDNA3-Dtk construct was electroporated into several

heterologous cell lines to demonstrate expression of Dtk. Electroporation, drug selection and isolation of Dtk-expressing clones for each cell line followed standard techniques (in M. Kriegler, "Gene Transfer and Expression - A Laboratory Manual", Stockton Press, New York 1990).

The Dtk construct was expressed in the factor-dependent cell lines FDC-P1,

BAF/3 and 32D, and in the NIH 3T3 cell line (all commercially available). The expression of Dtk in these cell lines has been ascertained at the level of RNA using standard techniques for the isolation of RNA and its detection using radiolabelled Dtk probes which are familiar to those experienced in the field (see Sambrook et al., "Molecular Cloning," Second Edition, supra vide).

D. Ligands

The invention also includes ligands that bind to the Dtks of the invention. The ligand may be a protein such as a growth factor that occurs naturally in a mammal, preferably the same mammal that produces the corresponding Dtk. The

growth factor may be isolated and purified, or be present on the surface of an

isolated population of cells, such as stromal cells.

The ligand may also be a molecule that does not occur naturally in a mammal. For example, antibodies raised against the receptors of the invention or against

anti-ligand antibodies mimic the shape of, and act as, ligands if they constitute the negative image of the receptor or anti-ligand antibody binding site.

Such antibodies may be polyclonal but are preferably monoclonal. Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256. 495- 497 (1975) and Campbell in "Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas" in Burdon et al. Eds,

Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well as by the recombinant DNA method described by Huse et al. in Science 246. 1275-1281 (1989).

In yet another form, the ligand may also be a non-protein molecule that acts as a ligand when it binds to, or otherwise comes into contact with, the receptor.

In addition, ligands may be of two functional types. The first functional type of ligand is a molecule which binds to the receptor and stimulates it in performing its normal function (a "stimulant ligand"). The second functional type of ligand is a molecule which binds to the receptor and inhibits or prevents it performing its normal function (an "antagonistic ligand").

Both types of ligand will find application in either therapeutic or prophylactic treatments as described below.

D.l Sources of Ligands

The strategy for isolating a ligand for the Dtks of the invention is based on the assumption that the ligand will either be a soluble, secreted protein or alternatively it will be membrane-bound or associated.

To screen for soluble ligands, conditioned media from a range of tumor cell lines and tissues can be used. Such cell lines are readily available from the American Type Culture Collection (ATCC) Rockville, Maryland, USA. Conditioned media is generated from these cell lines using a variety of culture and induction protocols. The cell lines are grown using standard tissue culture techniques which are detailed by ATCC for each cell line. Conditioned medium from tissues is generated by growing minced tissue fragments in culture medium for a defined time period.

To screen for membrane-associated ligands a different approach is taken. Cell lines in which from tissues which are in close proximity to those cells or tissues which have been shown to express the Dtk receptor are used. This approach is based on the likelihood of close cell-to-cell contact between receptor-expressing cells and ligand-expressing cells. An example of this is in the testis where Sertoli cells express the receptor, while germ cells are considered a likely source of

membrane-bound ligand. A further example in the brain would be where one

type of neuron expresses the receptor, while microglial cells or another non-

neuronal brain cell are considered likely to express the ligand.

D.2 Ligand Screening Procedures

In illustrating the screening procedures, reference will be made to murine Dtk as representative of the Dtks of the invention. Equivalent procedures can of course be employed in screening protocols using other mammalian Dtks such as human Dtk or the extracellular receptor domains of such Dtks.

Two approaches are followed to screen for the ligand for murine Dtk. If the ligand is soluble, assays which utilise either growth responses or changes in tyrosine phosphorylation will be used. Alternatively, if the ligand is membrane- bound, ligand-expressing cells will be detected using a Dtk-tag protein system whereby the extracellular domain of Dtk is fused with sequence encoding part of the human immunoglobulin molecule, such as the Fc region or the μ chain. The tag can then be detected using reagents which bind to the tag, such as Protein A- alkaline phosphatase or Protein A-radioiodine¹²⁵.

D.3 Soluble ligand

To detect soluble ligand in the media conditioned by tumor cell lines or tissues, a range of concentrations of this media are added to one of the factor-dependent cell lines described above, that have been transfected with, and express the Dtk receptor. These cell lines are routinely maintained in interleukin-3 containing tissue culture medium. By withdrawing this medium and adding sources of potential ligand for Dtk, a growth response will be sought that is mediated via the introduced Dtk receptor. This response can be detected using the uptake of radiolabelled thymidine and counting this uptake by liquid scintillation spectroscopy. These techniques are standard for those familiar in the art (see

Kriegler (supra); and Crosier et al, Proc. Νatl. Acad. Sci. USA 88: 7744-8 (1991)).

An alternative detection system for ligands contained in tumor cell line conditioned medium uses the Dtk-expressing ΝIH 3T3 cell line as an indicator system, in conjunction with monitoring alterations in tyrosine phosphorylation of the Dtk receptor. Conditioned medium that contains the ligand for Dtk will trigger activation of the receptor which in turn is reflected in the phosphorylation status of the receptor. The system uses standard techniques whereby the ΝIH 3T3 cells are incubated with conditioned medium, cell lysates produced which in turn are immunoprecipitated with an anti-murine Dtk polyclonal antibody, proteins are resolved on SDS-PAGE gels, followed by transfer to nitrocellulose filters and subsequent Western blotting with an anti-phosphotyrosine antibody and detection using enhanced chemiluminescence techmques. These techniques are standard protein biochemistry methods (see B. Sefton and T. Hunter (eds), "Methods in Enzymology," vol 200 and 201, 1990; and Amersham, Manufacturer's protocols for

ECL techniques). The expected result with this technique would be that potential ligand-containing media would stimulate increased tyrosine phosphorylation, compared with background levels detected in these cells. D.4 Membrane-bound ligand

Screening for membrane-bound or associated ligands for the Dtk receptor relies

on the use of a Dtk-tag fusion protein detection system. The extracellular domain

of the Dtk receptor is fused in-frame to the Fc region of human immunoglobulin (IgG) or to part of the human μ chain of IgM. This procedure follows that described by Goodwin et al., Cell 73: 447-456 (1993). The fusion protein is produced by transfecting the fused genes contained within the expression pEDδ c vector into COS cells. The fusion protein is purified on Protein A-Sepharose columns (Pharmacia). The Dtk-tag fusion proteins are biotinylated using sulfosuccinimiddyl-6-biotinamido)-hexanoate (Pierce Chemicals) according to the manufacturer's procedures. Alternatively, FITC-conjugated Dtk-tag fusion protein is generated by conjugating the fusion protein to FITC using standard techniques (see Suda et al.. Cell 75: 169-1178, 1993).

The Dtk-tag fusion protein is used to screen for the expression of bound Dtk protein on tumor cell lines using flow cytometric techniques. The techniques used for the labelling of cells and flow cytometric analysis follow those described by Mosley et al., Cell 59: 335-348 (1989). Tumor cells are labelled on ice with the biotinylated Dtk-fusion protein using avidin-FITC, or the FITC-labelled protein is used directly in FACS analysis. The screening procedure is aimed at detecting a cell line that produces a signal above background with the Dtk-tag fusion protein, compared with an unrelated receptor-tag fusion protein. Sequential FACS sorting of Dtk ligand-expressing cells is undertaken to generate a high Dtk ligand- expressing tumor cell subline which can be used for the generation of a cDΝA expression library (for an overview of this strategy see Wong in Genetic Engineering Vol. 12. ed by J K Setlow, 1990).

E Expression Cloning of the Dtk Ligand E.l Construction of an expression library

A random-primed expression library is constructed from poly(A)⁺ mRΝA isolated from the cell line or tissue demonstrated to give a positive signal in either the growth assay, phosphorylation assay or Dtk-tag fusion protein assay outlined

above. The techniques used for construction of the expression library are standard

procedures for those experienced in the field (see McMahon et al, EMBO J. 10,

2821-2832, 1991; and Kriegler (supra)).

E.2 Cloning of the murine or human Dtk ligand

The expression library constructed from the cell line or tissue is screened by transfecting pools of cDΝAs into COS cells using standard techniques (see

Sambrook et al, supra). Two approaches are used to detect positive pools, depending on whether there has been evidence for either a soluble form of ligand or a membrane-bound form of ligand.

Soluble forms: COS supernatants are screened in the detection systems outlined above for soluble ligand forms. COS cells are grown in 10 cm plates using standard tissue culture techniques.

Membrane-bound forms: COS cells are grown in LabTech (Νunc) chambers and positive pools are detected by using the binding of Dtk-tag fusion protein to the COS cells, followed by detection with either a Protein A-horseradish peroxidase enzymatic reaction or Protein A-¹²⁵I binding and subsequent autoradiography.

Procedures for the breaking down of cDΝA pools, subsequent sib selection and the isolation of single cDΝA clones are outlined in Sambrook et al., (supra) and

Wong (supra). Sequence analysis of single cDΝAs follows standard techniques. Once a single cDΝA clone is isolated this is transfected into COS cells or into CHO cells for large scale production of protein using standard procedures.

F Application of Ligands for the Dtks of the Invention

The types of ligand discussed above can be employed in two distinctive methods in accordance with this invention.

The first such method is a method of stimulating the proliferation, differentiation and/or survival of a cell expressing a Dtk of the invention. This stimulation, which can occur in vivo or ex vivo, involves contacting the cell with an effective amount of the ligand.

The ability of a ligand according to the invention to stimulate cells such as stem cells which express the Dtk of the invention has important therapeutic applications. Such applications include medically treating mammals, including humans, whose stem cells do not sufficiently undergo self-renewal. Examples of such medical problems which can be treated in this way include those that occur when defects in haematopoietic stem cells or their related growth factors depress the number of blood cells, leading to disorders such as aplastic anaemia. The treatment of bone marrow damage resulting from cancer chemotherapy and radiation is another example of a medical problem that could be treated in this way.

The method can also be applied in stimulating the proliferation, differentiation and/or survival of mammalian fetal or adult neuronal cells or cells that form part of the central nervous system. Again, this has important therapeutic applications. Such applications include treating mammals, including humans, for inherited or degenerative disorders of the central nervous system. An additional application is

the treatment of individuals with central nervous system trauma, for example, spinal cord trauma resulting from either crushing or asphyxia.

Yet a further therapeutic application for the ligands of the invention is in sports medicine, particularly in the treatment of muscle injuries. The Dtk of the invention is abundantly expressed on myoblast cells but not on mature muscle cells. Application of the ligand will stimulate myoblast cell proliferation and differentiation, leading to muscle repair.

In terms of ex vivo applications, the method has implications for gene therapy. In gene therapy genes are inserted into host cells (such as haematopoietic stem cells and myoblasts) and the expression of the gene regulated by either an endogenous or an exogenous promoter. However, it is often difficult to maintain growth and survival of these cells ex vivo while they are being manipulated for the insertion of foreign genes. Therefore, as the Dtk of the invention is expressed on haematopoietic stem cells and myoblasts, the ligand has a direct application in stimulating the growth, proliferation or simple survival of their cells during the manipulative process.

The second distinct method of the invention is a method of inhibiting the function of the Dtk of the invention. This method, which would normally be applied in vivo for both prophylactic and therapeutic applications, involves contacting the receptor with a ligand which blocks or prevents stimulation of the receptor (an

antagonist ligand).

In terms of prophylaxis, such a method has specific application to the Sertoli cells of the testis, which abundantly express the receptor. Due to the involvement of these Sertoli cells in male fertility, contacting the receptors with an antagonistic ligand has a potential application in the control of male fertility including in male contraception.

A potential therapeutic application of contacting cells expressing the Dtk of the invention with an antagonistic ligand is in anti-tumour therapy. This potential application arises from the growing understanding of the role sometimes played by RTKs in tumour formation.

G Therapeutic Applications of Soluble Receptors

The extracellular receptor domain of the invention as described above also have potential therapeutic applications. Such applications are in a method of treating a disease, syndrome or condition caused or mediated by an excess of a ligand of the invention (whether stimulant or antagonistic). In this method, the extracellular receptor domain of the Dtk in a soluble form can be used as a molecular "sponge" or "sink" to remove the excess of the ligand or at least to block its activity.

H Functional Equivalents

The invention includes functional equivalents of the Dtks, receptor domains, nucleic acid molecules and ligands described above.

The Dtks, extracellular receptor domains and ligands are or include proteins. A protein is considered a functional equivalent of another protein for a specific function if the equivalent protein is immunologically cross-reactive with, and has the same function as, the original protein. The equivalent may, for example, be a fragment of the protein, or a substitution, addition or deletion mutant of the protein.

For example, it is possible to substitute amino acids in a sequence with equivalent amino acids using conventional techniques. Groups of amino acids known normally to be equivalent are:

(a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G); (b) Asn(Ν) Asp(D) Glu(E) Gln(Q);

(c) His(H) Arg(R) Lys(K);

(d) Met(M) Leu(L) Ile(I) Val(V); and

(e) Phe(F) Tyr(Y) Trp(W). Substitutions, additions and/or deletions in the receptors and ligands may be made as long as the resulting equivalent receptors and ligands are immunologically

cross-reactive with, and have the same function as, the native receptors and

ligands.

The equivalent receptors and ligands will normally have substantially the same amino acid sequence as the native receptors and ligands. An amino acid sequence

that is substantially the same as another sequence, but that differs from the other sequence by means of one or more substitutions, additions and/or deletions is considered to be an equivalent sequence. Preferably, less than 25%, more preferably less than 10%, and most preferably less than 5% of the number of amino acid residues in the amino acid sequence of the native receptors and ligands are substituted for, added to, or deleted from.

Equivalent nucleic acid molecules include nucleic acid sequences that encode equivalent receptors and ligands as defined above. Equivalent nucleic acid molecules also include nucleic acid sequences that, due to the degeneracy of the nucleic acid code, differ from native nucleic acid sequences in ways that do not affect the corresponding amino acid sequences.

Those persons skilled in the art will of course appreciate that the above

description is provided by way of example only and that the invention is limited only by the lawful scope of the appended claims. SEQUEΝCE LISΗΝG

(1) GENERAL INFORMAΗON:

(1) AUCKLAND UNISERVICES LIMITED, a duly incoφorated New Zealand company c/- The University of Auckland, 58 Symonds Street, Auckland, New Zeaalnd.

(2) TITLE OF INVENTION: Developmental Tyrosine Kinases and

their Ligands.

(3) NUMBER OF SEQUENCES: 12

(4) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: A J PARK & SON

(B) STREET: HUDDART PARKER BUILDING, POST OFFICE SQUARE

(C) CITY: P O BOX 949, WELLINGTON

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(D) SOFTWARE: WORD PERFECT 5.1 FOR DOS (6) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 16-FEBRUARY 1994

(C) CLASSIFICATION

(7) ATTORNEY/ AGENT INFORMATION: (A) NAME: BENNETT, MICHAEL R.

(8) TELECOMMUNICATION INFORMATION

(A) TELEPHONE: (64 4) 473 8278

(B) TELEFAX: (64 4) 472 3358

(2) INFORMATION FOR SEQUENCE ID NO. 1:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 874 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: PROTEIN

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 1:

Met Gly Trp Pro Gly Leu Arg Pro Leu Leu Leu Ala Gly 13

Leu Ala Ser Leu Leu Leu Pro Gly Ser Ala Ala Ala Gly 26

Leu Lys Leu Met Gly Ala Pro Val Lys Met Thr Val Ser 39

Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val Glu Gly 52

Met Glu Asp Pro Asp He His Trp Met Lys Asp Gly Thr 65

Val Val Gin Asn Ala Ser Gin Val Ser He Ser He Ser 78 Glu His Ser Trp He Gly Leu Leu Ser Leu Lys Ser Val 91

Glu Arg Ser Asp Ala Gly Leu Tyr Trp Cys Gin Val Lys 104

Asp Gly Glu Glu Thr Lys He Ser Gin Ser Val Trp Leu 117

Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu Pro Lys 130

Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin Leu Ser 143

Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr He Tyr 156

Trp Trp Arg Gly Leu Thr Lys Val Gly Gly Pro Ala Pro 169

Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr Gin Arg 182

Thr Glu Phe Ser Cys Glu Ala Arg Asn He Lys Gly Leu 195

Ala Thr Ser Arg Pro Ala He Val Arg Leu Gin Ala Pro 208

Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr He Ser 221

Ser Tyr Asn Ala Ser Val Ala Trp Val Pro Gly Ala Asp 234

Gly Leu Ala Leu Leu His Ser Cys Thr Val Gin Val Ala 247

His Ala Pro Gly Glu Trp Glu Ala Leu Ala Val Val Val 260

Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu Ala 273

Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn 286

Ala Leu Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe 299

Gin Thr Lys Gly Leu Ala Pro Ala Arg Ala Pro Gin Asn 312

Phe His Ala He Arg Thr Asp Ser Gly Leu He Leu Glu 325

Trp Glu Glu Val He Pro Glu Asp Pro Gly Glu Gly Pro 338

Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Glu Asn Gly 351

Thr Gin Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn 364

Leu Thr Asp Trp Asp Pro Gin Lys Asp Leu He Leu Arg 377

Val Cys Ala Ser Asn Ala He Gly Asp Gly Pro Trp Ser 390

Gin Pro Leu Val Val Ser Ser His Asp His Ala Gly Arg 403

Gin Gly Pro Pro His Ser Arg Thr Ser Trp Val Pro Val 416

Val Leu Gly Val Leu Thr Ala Leu He Thr Ala Ala Ala 429

Leu Ala Leu He Leu Leu Arg Lys Arg Arg Lys Glu Thr 442

Arg Phe Gly Gin Ala Phe Asp Ser Val Met Ala Arg Gly 455

Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser Phe Asn 468

Arg Glu Arg Pro Glu Arg He Glu Ala Thr Leu Asp Ser 481

Leu Gly He Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp 494

Val Leu He Pro Glu Gin Gin Phe Thr Leu Gly Arg Met 507

Leu Gly Lys Gly Glu Phe Gly Ser Val Arg Glu Ala Gin 520 Leu Lys Gin Glu Asp Gly Ser Phe Val Lys Val Ala Val 533

Lys Met Leu Lys Ala Asp He He Ala Ser Ser Asp He 546

Glu Glu Phe leu Arg Glu Ala Ala Cys Met Lys Glu Phe 559

Asp His Pro His Val Ala Lys Leu Val Gly Val Ser Leu 572

Arg Ser Arg Ala Lys Gly Arg Leu Pro He Pro Met Val 585

He Leu Pro Phe Met Lys His Gly Asp Leu His Ala Phe 598

Leu Leu Ala Ser Arg He Gly Glu Asn Pro Phe Asn Leu 611

Pro Leu Gin Thr Leu Val Arg Phe Met Val Asp He Ala 624

Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe He His 637

Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Ala Glu Asp 650

Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Arg Lys 663

He Tyr Ser Gly Asp Tyr Tyr Arg Gin Gly Cys Ala Ser 676

Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu Ala 689

Asp Asn Leu Tyr Thr Val His Ser Asp Val Trp Ala Phe 702

Gly Val Thr Met Trp Glu He Met Thr Arg Gly Gin Thr 715

Pro Tyr Ala Gly He Glu Asn Ala Glu He Tyr Asn Tyr 728

Leu He Gly Gly Asn Arg Leu Lys Gin Pro Pro Glu Cys 741

Met Glu Glu Val Tyr Asp Leu Met Tyr Gin Cys Trp Ser 754

Ala Asp Pro Lys Gin Arg Pro Ser Phe Thr Cys Leu Arg 767

Met Glu Leu Glu Asn He Leu Gly His Leu Ser Val Leu 780

Ser Thr Ser Gin Asp Pro Leu Tyr He Asn He Glu Arg 793

Ala Glu Gin Pro Thr Glu Ser Gly Ser Pro Glu Leu His 806

Cys Gly Glu Arg Ser Ser Ser Glu Ala Gly Asp Gly Ser 819

Gly Val Gly Ala Val Gly Gly He Pro Ser Asp Ser Arg 832

Tyr He Phe Ser Pro Gly Gly Leu Ser Glu Ser Pro Gly 845

Gin Leu Glu Gin Gin Pro Glu Ser Pro Leu Asn Glu Asn 858

Gin Arg Leu Leu Leu Leu Gin Gin Gly Leu Leu Pro His 871

Ser Ser Cys 874

(3) INFORMATION FOR SEQUENCE ID NO. 2:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 850 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) TOPOLOGY: LINEAR (2) MOLECULE TYPE: PROTEIN

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 2:

Ala Gly 2

Leu Lys Leu Met Gly Ala Pro Val Lys Met Thr Val Ser 15

Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val Glu Gly 28

Met Glu Asp Pro Asp He His Trp Met Lys Asp Gly Thr 41

Val Val Gin Asn Ala Ser Gin Val Ser He Ser He Ser 54

Glu His Ser Trp He Gly Leu Leu Ser Leu Lys Ser Val 67

Glu Arg Ser Asp Ala Gly Leu Tyr Trp Cys Gin Val Lys 80

Asp Gly Glu Glu Thr Lys He Ser Gin Ser Val Trp Leu 93

Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu Pro Lys 106

Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin Leu Ser 119

Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr He Tyr 132

Trp Trp Arg Gly Leu Thr Lys Val Gly Gly Pro Ala Pro 145

Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr Gin Arg 158

Thr Glu Phe Ser Cys Glu Ala Arg Asn He Lys Gly Leu 171

Ala Thr Ser Arg Pro Ala He Val Arg Leu Gin Ala Pro 184

Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr He Ser 197

Ser Tyr Asn Ala Ser Val Ala Trp Val Pro Gly Ala Asp 210

Gly Leu Ala Leu Leu His Ser Cys Thr Val Gin Val Ala 223

His Ala Pro Gly Glu Trp Glu Ala Leu Ala Val Val Val 236

Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu Ala 249

Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn 262

Ala Leu Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe 275

Gin Thr Lys Gly Leu Ala Pro Ala Arg Ala Pro Gin Asn 288

Phe His Ala He Arg Thr Asp Ser Gly Leu He Leu Glu 301

Trp Glu Glu Val He Pro Glu Asp Pro Gly Glu Gly Pro 314

Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Glu Asn Gly 327

Thr Gin Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn 340

Leu Thr Asp Trp Asp Pro Gin Lys Asp Leu He Leu Arg 353

Val Cys Ala Ser Asn Ala He Gly Asp Gly Pro Trp Ser 366

Gin Pro Leu Val Val Ser Ser His Asp His Ala Gly Arg 379

Gin Gly Pro Pro His Ser Arg Thr Ser Trp Val Pro Val 392

Val Leu Gly Val Leu Thr Ala Leu He Thr Ala Ala Ala 405 Leu Ala Leu He Leu Leu Arg Lys Arg Arg Lys Glu Thr 418

Arg Phe Gly Gin Ala Phe Asp Ser Val Met Ala Arg Gly 431

Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser Phe Asn 444

Arg Glu Arg Pro Glu Arg He Glu Ala Thr Leu Asp Ser 457

Leu Gly He Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp 470

Val Leu He Pro Glu Gin Gin Phe Thr Leu Gly Arg Met 483

Leu Gly Lys Gly Glu Phe Gly Ser Val Arg Glu Ala Gin 496

Leu Lys Gin Glu Asp Gly Ser Phe Val Lys Val Ala Val 509

Lys Met Leu Lys Ala Asp He He Ala Ser Ser Asp He 522

Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe 535

Asp His Pro His Val Ala Lys Leu Val Gly Val Ser Leu 548

Arg Ser Arg Ala Lys Gly Arg Leu Pro He Pro Met Val 561

He Leu Pro Phe Met Lys His Gly Asp Leu His Ala Phe 574

Leu Leu Ala Ser Arg He Gly Glu Asn Pro Phe Asn Leu 587

Pro Leu Gin Thr Leu Val Arg Phe Met Val Asp He Ala 600

Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe He His 613

Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Ala Glu Asp 626

Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Arg Lys 639

He Tyr Ser Gly Asp Tyr Tyr Arg Gin Gly Cys Ala Ser 652

Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu Ala 665

Asp Asn Leu Tyr Thr Val His Ser Asp Val Trp Ala Phe 678

Gly Val Thr Met Trp Glu He Met Thr Arg Gly Gin Thr 691

Pro Tyr Ala Gly He Glu Asn Ala Glu He Tyr Asn Tyr 704

Leu He Gly Gly Asn Arg Leu Lys Gin Pro Pro Glu Cys 717

Met Glu Glu Val Tyr Asp Leu Met Tyr Gin Cys Trp Ser 730

Ala Asp Pro Lys Gin Arg Pro Ser Phe Thr Cys Leu Arg 743

Met Glu Leu Glu Asn He Leu Gly His Leu Ser Val Leu 756

Ser Thr Ser Gin Asp Pro Leu Tyr He Asn He Glu Arg 769

Ala Glu Gin Pro Thr Glu Ser Gly Ser Pro Glu Leu His 782

Cys Gly Glu Arg Ser Ser Ser Glu Ala Gly Asp Gly Ser 795

Gly Val Gly Ala Val Gly Gly He Pro Ser Asp Ser Arg 808

Tyr He Phe Ser Pro Gly Gly Leu Ser Glu Ser Pro Gly 821

Gin Leu Glu Gin Gin Pro Glu Ser Pro Leu Asn Glu Asn 834

Gin Arg Leu Leu Leu Leu Gin Gin Gly Leu Leu Pro His 847 Ser Ser Cys 850

(4) IΝFORMAΗOΝ FOR SEQUENCE ID NO. 3:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 876 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: PROTEIN

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 3:

Met Gly Arg Pro Gly Leu Pro Pro Leu Pro Leu Pro Pro 13

Pro Pro Arg Leu Gly Leu Leu Leu Ala Glu Ser Ala Ala 26

Ala Gly Leu Lys Leu Met Gly Ala Pro Val Lys Leu Thr 39

Val Ser Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val 52

Glu Gly Met Glu Glu Pro Asp He Gin Trp Val Lys Asp 65

Gly Ala Val Val Gin Asn Leu Asp Gin Leu Tyr He Pro 78

Val Ser Glu Gin His Trp He Gly Phe Leu Ser Leu Lys 91

Ser Val Glu Arg Ser Asp Ala Gly Arg Tyr Trp Cys Gin 104

Val Glu Asp Gly Gly Glu Thr Glu He Ser Gin Pro Val 117

Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu 130

Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin 143

Leu Ser Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr 156

He Val Trp Trp Arg Gly Thr Thr Lys He Gly Gly Pro 169

Ala Pro Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr 182

Gin Ser Thr Met Phe Ser Cys Glu Ala His Asn Leu Lys 195

Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His Leu Gin 208

Ala Leu Pro Ala Ala Pro Phe Asn He Thr Val Thr Lys 221

Leu Ser Ser Ser Asn Ala Ser Val Ala Trp Met Pro Gly 234

Ala Asp Gly Arg Ala Leu Leu Gin Ser Cys Thr Val Gin 247

Val Thr Gin Ala Pro Gly Gly Trp Glu Val Leu Ala Val 260

Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asp 273

Leu Val Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys 286

Ala Asn Ala Leu Gly Pro Ser Pro Tyr Ala Asp Trp Val 299

Pro Phe Gin Thr Lys Gly Leu Ala Pro Ala Ser Ala Pro 312

Gin Asn Leu His Ala He Arg Thr Asp Ser Gly Leu He 325 Leu Glu Trp Glu Glu Val He Pro Glu Ala Pro Leu Glu 338

Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Asp 351

Asn Gly Thr Gin Asp Glu Leu Thr Val Glu Gly Thr Arg ^64

Ala Asn Leu Thr Gly Trp Asp Pro Gin Lys Asp Leu He 377

Val Arg Val Cys Val Ser Asn Ala Val Gly Cys Gly Pro 390

Trp Ser Gin Pro Leu Val Val Ser Ser His Asp Arg Ala 403

Gly Gin Gin Gly Pro Pro His Ser Arg Thr Ser Trp Val 416

Pro Val Val Leu Gly Val Leu Thr Ala Leu Val Thr Ala 429

Ala Ala Leu Ala Leu He Leu Leu Arg Lys Arg Arg Lys 442

Glu Thr Arg Phe Gly Gin Ala Phe Asp Ser Val Met Ala 455

Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser 468

Phe Asn Arg Glu Arg Pro Glu Arg He Glu Ala Thr Leu 481

Asp Ser Leu Gly He Ser Asp Glu Leu Lys Glu Lys Leu 494

Glu Asp Val Leu He Pro Glu Gin Gin Phe Thr Leu Gly 507

Arg Met Leu Gly Lys Gly Glu Phe Gly Ser Val Arg Glu 520

Ala Gin Leu Lys Gin Glu Asp Gly Ser Phe Val Lys Val 533

Ala Val Lys Met Leu Lys Ala Asp He He Ala Ser Ser 546

Asp He Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys 559

Glu Phe Asp His Pro His Val Ala Lys Leu Val Gly Val 572

Ser Leu Arg Ser Arg Ala Lys Gly Arg Leu Pro He Pro 585

Met Val He Leu Pro Phe Met Lys His Gly Asp Leu His 598

Ala Phe Leu Leu Ala Ser Arg He Gly Glu Asn Pro Phe 611

Asn Leu Pro Leu Gin Thr Leu He Arg Phe Met Val Asp 624

He Ala Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe 637

He His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Ala 650

Glu Asp Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser 663

Arg Lys He Tyr Ser Gly Asp Tyr Tyr Arg Gin Gly Cys 676

Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser 689

Leu Ala Asp Asn Leu Tyr Thr Val Gin Ser Asp Val Trp 702

Ala Phe Gly Val Thr Met Trp Glu He Met Thr Arg Gly 715

Gin Thr Pro Tyr Ala Gly He Glu Asn Ala Glu He Tyr 728

Asn Tyr Leu He Gly Gly Asn Arg Leu Lys Gin Pro Pro 741

Glu Cys Met Glu Asp Val Tyr Asp Leu Met Tyr Gin Cys 754

Trp Ser Ala Asp Pro Lys Gin Arg Pro Ser Phe Thr Cys 767 Leu Arg Met Glu Leu Glu Asn He Leu Gly Gin Leu Ser 780

Val Leu Ser Ala Ser Gin Asp Pro Leu Tyr He Asn He 793

Glu Arg Ala Glu Glu Pro Thr Val Gly Gly Ser Leu Glu 806

Leu Pro Gly Arg Asp Gin Pro Tyr Ser Gly Ala Gly Asp 819

Gly Ser Gly Met Gly Ala Val Gly Gly Thr Pro Ser Asp 832

Cys Arg Tyr He Leu Thr Pro Gly Gly Leu Ala Glu Gin 845

Pro Gly Gin Ala Glu His Gin Pro Glu Ser Pro Leu Asn 858

Glu Thr Gin Arg Leu Leu Leu Leu Gin Gin Gly Leu Leu 871

Pro His Ser Ser Cys 876

(5) INFORMATION FOR SEQUENCE ID NO. 4:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 850 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: PROTEIN

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 4:

Ala Gly Leu Lys Leu Met Gly Ala Pro Val Lys Leu Thr 13

Val Ser Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val 26

Glu Gly Met Glu Glu Pro Asp He Gin Trp Val Lys Asp 39

Gly Ala Val Val Gin Asn Leu Asp Gin Leu Tyr He Pro 52

Val Ser Glu Gin His Trp He Gly Phe Leu Ser Leu Lys 65

Ser Val Glu Arg Ser Asp Ala Gly Arg Tyr Trp Cys Gin 78

Val Glu Asp Gly Gly Glu Thr Glu He Ser Gin Pro Val 91

Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu 104

Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin 117

Leu Ser Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr 130

He Val Trp Trp Arg Gly Thr Thr Lys He Gly Gly Pro 143

Ala Pro Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr 156

Gin Ser Thr Met Phe Ser Cys Glu Ala His Asn Leu Lys 169

Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His Leu Gin 182

Ala Leu Pro Ala Ala Pro Phe Asn He Thr Val Thr Lys 195

Leu Ser Ser Ser Asn Ala Ser Val Ala Trp Met Pro Gly 208

Ala Asp Gly Arg Ala Leu Leu Gin Ser Cys Thr Val Gin 221

Val Thr Gin Ala Pro Gly Gly Trp Glu Val Leu Ala Val 234 Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asp 247

Leu Val Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys 260

Ala Asn Ala Leu Gly Pro Ser Pro Tyr Ala Asp Trp Val 273

Pro Phe Gin Thr Lys Gly Leu Ala Pro Ala Ser Ala Pro 286

Gin Asn Leu His Ala He Arg Thr Asp Ser Gly Leu He 299

Leu Glu Trp Glu Glu Val He Pro Glu Ala Pro Leu Glu 312

Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Asp 325

Asn Gly Thr Gin Asp Glu Leu Thr Val Glu Gly Thr Arg 338

Ala Asn Leu Thr Gly Trp Asp Pro Gin Lys Asp Leu He 351

Val Arg Val Cys Val Ser Asn Ala Val Gly Cys Gly Pro 364

Trp Ser Gin Pro Leu Val Val Ser Ser His Asp Arg Ala 377

Gly Gin Gin Gly Pro Pro His Ser Arg Thr Ser Trp Val 390

Pro Val Val Leu Gly Val Leu Thr Ala Leu Val Thr Ala 403

Ala Ala Leu Ala Leu He Leu Leu Arg Lys Arg Arg Lys 416

Glu Thr Arg Phe Gly Gin Ala Phe Asp Ser Val Met Ala 429

Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser 442

Phe Asn Arg Glu Arg Pro Glu Arg He Glu Ala Thr Leu 455

Asp Ser Leu Gly He Ser Asp Glu Leu Lys Glu Lys Leu 468

Glu Asp Val Leu He Pro Glu Gin Gin Phe Thr Leu Gly 481

Arg Met Leu Gly Lys Gly Glu Phe Gly Ser Val Arg Glu 494

Ala Gin Leu Lys Gin Glu Asp Gly Ser Phe Val Lys Val 507

Ala Val Lys Met Leu Lys Ala Asp He He Ala Ser Ser 520

Asp He Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys 533

Glu Phe Asp His Pro His Val Ala Lys Leu Val Gly Val 546

Ser Leu Arg Ser Arg Ala Lys Gly Arg Leu Pro He Pro 559

Met Val He Leu Pro Phe Met Lys His Gly Asp Leu His 572

Ala Phe Leu Leu Ala Ser Arg He Gly Glu Asn Pro Phe 585

Asn Leu Pro Leu Gin Thr Leu He Arg Phe Met Val Asp 598

He Ala Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe 611

He His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Ala 624

Glu Asp Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser 637

Arg Lys He Tyr Ser Gly Asp Tyr Tyr Arg Gin Gly Cys 650

Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser 663

Leu Ala Asp Asn Leu Tyr Thr Val Gin Ser Asp Val Trp 676 Ala Phe Gly Val Thr Met Trp Glu He Met Thr Arg Gly 689

Gin Thr Pro Tyr Ala Gly He Glu Asn Ala Glu He Tyr 702

Asn Tyr Leu He Gly Gly Asn Arg Leu Lys Gin Pro Pro 715

Glu Cys Met Glu Asp Val Tyr Asp Leu Met Tyr Gin Cys 728

Trp Ser Ala Asp Pro Lys Gin Arg Pro Ser Phe Thr Cys 741

Leu Arg Met Glu Leu Glu Asn He Leu Gly Gin Leu Ser 754

Val Leu Ser Ala Ser Gin Asp Pro Leu Tyr He Asn He 767

Glu Arg Ala Glu Glu Pro Thr Val Gly Gly Ser Leu Glu 780

Leu Pro Gly Arg Asp Gin Pro Tyr Ser Gly Ala Gly Asp 793

Gly Ser Gly Met Gly Ala Val Gly Gly Thr Pro Ser Asp 806

Cys Arg Tyr He Leu Thr Pro Gly Gly Leu Ala Glu Gin 819

Pro Gly Gin Ala Glu His Gin Pro Glu Ser Pro Leu Asn 832

Glu Thr Gin Arg Leu Leu Leu Leu Gin Gin Gly Leu Leu 845

Pro His Ser Ser Cys 850

(6) INFORMATION FOR SEQUENCE ID NO. 5:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 386 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: PROTEIN

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 5:

Ala Gly 2

Leu Lys Leu Met Gly Ala Pro Val Lys Met Thr Val Ser 15

Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val Glu Gly 28

Met Glu Asp Pro Asp He His Trp Met Lys Asp Gly Thr 41

Val Val Gin Asn Ala Ser Gin Val Ser He Ser He Ser 54

Glu His Ser Trp He Gly Leu Leu Ser Leu Lys Ser Val 67

Glu Arg Ser Asp Ala Gly Leu Tyr Trp Cys Gin Val Lys 80

Asp Gly Glu Glu Thr Lys He Ser Gin Ser Val Trp Leu 93

Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu Pro Lys 106

Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin Leu Ser 119

Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr He Tyr 132

Trp Trp Arg Gly Leu Thr Lys Val Gly Gly Pro Ala Pro 145

Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr Gin Arg 158 Thr Glu Phe Ser Cys Glu Ala Arg Asn He Lys Gly Leu 171

Ala Thr Ser Arg Pro Ala He Val Arg Leu Gin Ala Pro 184

Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr He Ser 197

Ser Tyr Asn Ala Ser Val Ala Trp Val Pro Gly Ala Asp 210

Gly Leu Ala Leu Leu His Ser Cys Thr Val Gin Val Ala 223

His Ala Pro Gly Glu Trp Glu Ala Leu Ala Val Val Val 236

Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu Ala 249

Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn 262

Ala Leu Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe 275

Gin Thr Lys Gly Leu Ala Pro Ala Arg Ala Pro Gin Asn 288

Phe His Ala He Arg Thr Asp Ser Gly Leu He Leu Glu 301

Trp Glu Glu Val He Pro Glu Asp Pro Gly Glu Gly Pro 314

Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Glu Asn Gly 327

Thr Gin Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn 340

Leu Thr Asp Trp Asp Pro Gin Lys Asp Leu He Leu Arg 353

Val Cys Ala Ser Asn Ala He Gly Asp Gly Pro Trp Ser 366

Gin Pro Leu Val Val Ser Ser His Asp His Ala Gly Arg 379

Gin Gly Pro Pro His Ser Arg 386

(7) INFORMATION FOR SEQUENCE ID NO. 6:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 386 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: PROTEIN

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 6:

Ala Gly Leu Lys Leu Met Gly Ala Pro Val Lys Leu Thr 13

Val Ser Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val 26

Glu Gly Met Glu Glu Pro Asp He Gin Trp Val Lys Asp 39

Gly Ala Val Val Gin Asn Leu Asp Gin Leu Tyr He Pro 52

Val Ser Glu Gin His Trp He Gly Phe Leu Ser Leu Lys 65

Ser Val Glu Arg Ser Asp Ala Gly Arg Tyr Trp Cys Gin 78

Val Glu Asp Gly Gly Glu Thr Glu He Ser Gin Pro Val 91

Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu 104

Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin 1 7 Leu Ser Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr 130

He Val Trp Trp Arg Gly Thr Thr Lys He Gly Gly Pro 143

Ala Pro Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr 156

Gin Ser Thr Met Phe Ser Cys Glu Ala His Asn Leu Lys 169

Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His Leu Gin 182

Ala Leu Pro Ala Ala Pro Phe Asn He Thr Val Thr Lys 195

Leu Ser Ser Ser Asn Ala Ser Val Ala Trp Met Pro Gly 208

Ala Asp Gly Arg Ala Leu Leu Gin Ser Cys Thr Val Gin 221

Val Thr Gin Ala Pro Gly Gly Trp Glu Val Leu Ala Val 234

Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asp 247

Leu Val Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys 260

Ala Asn Ala Leu Gly Pro Ser Pro Tyr Ala Asp Trp Val 273

Pro Phe Gin Thr Lys Gly Leu Ala Pro Ala Ser Ala Pro 286

Gin Asn Leu His Ala He Arg Thr Asp Ser Gly Leu He 299

Leu Glu Trp Glu Glu Val He Pro Glu Ala Pro Leu Glu 312

Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Asp 325

Asn Gly Thr Gin Asp Glu Leu Thr Val Glu Gly Thr Arg 338

Ala Asn Leu Thr Gly Trp Asp Pro Gin Lys Asp Leu He 351

Val Arg Val Cys Val Ser Asn Ala Val Gly Cys Gly Pro 364

Trp Ser Gin Pro Leu Val Val Ser Ser His Asp Arg Ala 377

Gly Gin Gin Gly Pro Pro His Ser Arg 386

(8) INFORMATION FOR SEQUENCE ID NO. 7:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3919 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 7:

GGCACGAGTGTGGAAGGAGCGCGGTGGCCCAGCCGCAGCCCCGGGGACTCCTCGCTGCTG 60

ACGGCGGTGGCCGCGGCTCTAGGCGGCCGCGGGTCCCGGACGCCCCGGCCGAGCGCCGCC 120

CCCCGCCCCTCCCGCGGGCCTCCCGCCCCTCCTCCGCCACCCTCCTCTCAGCGCTCGCGG 180

GCCGGGCCCGGCATGGTGCGGCGTCGCCGCCGATGGCGCTGAGGCGGAGC 230

Met Gly Trp Pro Gly Leu Arg Pro Leu Leu Leu Ala Gly

ATG GGG TGG CCG GGG CTC CGG CCG CTG CTG CTG GCG GGA 269 Leu Ala Ser Leu Leu Leu Pro Gly Ser Ala Ala Ala Gly

CTG GCT TCT CTG CTG CTC CCC GGG TCT GCG GCC GCA GGC 308

Leu Lys Leu Met Gly Ala Pro Val Lys Met Thr Val Ser

CTG AAG CTC ATG GGC GCC CCA GTG AAG ATG ACC GTG TCT 347

Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val Glu Gly

CAG GGG CAG CCA GTG AAG CTC AAC TGC AGC GTG GAG GGG 386

Met Glu Asp Pro Asp He His Trp Met Lys Asp Gly Thr

ATG GAG GAC CCT GAC ATC CAC TGG ATG AAG GAT GGC ACC 425

Val Val Gin Asn Ala Ser Gin Val Ser He Ser He Ser

GTG GTC CAG AAT GCA AGC CAG GTG TCC ATC TCC ATC AGC 464

Glu His Ser Trp He Gly Leu Leu Ser Leu Lys Ser Val

GAG CAC AGC TGG ATT GGC TTA CTC AGC CTA AAG TCA GTG 503

Glu Arg Ser Asp Ala Gly Leu Tyr Trp Cys Gin Val Lys

GAG CGG TCT GAT GCT GGC CTG TAC TGG TGC CAG GTG AAG 542

Asp Gly Glu Glu Thr Lys He Ser Gin Ser Val Trp Leu

GAT GGG GAG GAA ACC AAG ATC TCT CAG TCA GTA TGG CTC 581

Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu Pro Lys

ACT GTC GAA GGT GTG CCA TTC TTC ACA GTG GAA CCA AAA 620

Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin Leu Ser

GAT CTG GCG GTG CCA CCC AAT GCC CCT TTT CAG CTG TCT 659

Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr He Tyr

TGT GAG GCT GTG GGT CCT CCA GAA CCC GTA ACC ATT TAC 698

Trp Trp Arg Gly Leu Thr Lys Val Gly Gly Pro Ala Pro

TGG TGG AGA GGA CTC ACT AAA GTT GGG GGA CCT GCT CCC 737

Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr Gin Arg

TCT CCC TCT GTT TTA AAT GTG ACA GGA GTG ACC CAG CGC 776

Thr Glu Phe Ser Cys Glu Ala Arg Asn He Lys Gly Leu

ACA GAG TTT TCT TGT GAA GCC CGC AAC ATA AAA GGC CTG 815

Ala Thr Ser Arg Pro Ala He Val Arg Leu Gin Ala Pro

GCC ACT TCC CGA CCA GCC ATT GTT CGC CTT CAA GCA CCG 854

Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr He Ser

CCT GCA GCT CCT TTC AAC ACC ACA GTA ACA ACG ATC TCC 893

Ser Tyr Asn Ala Ser Val Ala Trp Val Pro Gly Ala Asp

AGC TAC AAC GCT AGC GTG GCC TGG GTG CCA GGT GCT GAC 932

Gly Leu Ala Leu Leu His Ser Cys Thr Val Gin Val Ala

GGC CTA GCT CTG CTG CAT TCC TGT ACT GTA CAG GTG GCA 971

His Ala Pro Gly Glu Trp Glu Ala Leu Ala Val Val Val

CAC GCC CCA GGA GAA TGG GAG GCC CTT GCT GTT GTG GTT 1010

Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu Ala

CCT GTG CCA CCT TTT ACC TGC CTG CTT CGG AAC TTG GCC 1049

Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn

CCT GCC ACC AAC TAC AGC CTT AGG GTG CGC TGT GCC AAT 1088

Ala Leu Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe

GCC TTG GGC CCT TCT CCC TAC GGC GAC TGG GTG CCC TTT 1127

Gin Thr Lys Gly Leu Ala Pro Ala Arg Ala Pro Gin Asn

CAG ACA AAG GGC CTA GCG CCA GCC AGA GCT CCT CAG AAT 1166 Phe His Ala He Arg Thr Asp Ser Gly Leu He Leu Glu

TTC CAT GCC ATT CGT ACC GAC TCA GGC CTT ATC CTG GAA 1205

Trp Glu Glu Val He Pro Glu Asp Pro Gly Glu Gly Pro

TGG GAA GAA GTG ATT CCT GAG GAC CCT GGG GAA GGC CCC 1244

Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Glu Asn Gly

CTA GGA CCT TAT AAG CTG TCC TGG GTC CAA GAA AAT GGA 1283

Thr Gin Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn

ACC CAG GAT GAG CTG ATG GTG GAA GGG ACC AGG GCC AAT 1322

Leu Thr Asp Trp Asp Pro Gin Lys Asp Leu He Leu Arg

CTG ACC GAC TGG GAT CCC CAG AAG GAC CTG ATT TTG CGT 1361

Val Cys Ala Ser Asn Ala He Gly Asp Gly Pro Trp Ser

GTG TGT GCC TCC AAT GCA ATT GGT GAT GGG CCC TGG AGT 1400

Gin Pro Leu Val Val Ser Ser His Asp His Ala Gly Arg

CAG CCA CTG GTG GTG TCT TCT CAT GAC CAT GCA GGG AGG 1439

Gin Gly Pro Pro His Ser Arg Thr Ser Trp Val Pro Val

CAG GGC CCT CCC CAC AGC CGC ACA TCC TGG GTG CCT GTG 1478

Val Leu Gly Val Leu Thr Ala Leu He Thr Ala Ala Ala

GTC CTG GGC GTG CTC ACC GCC CTG ATC ACA GCT GCT GCC 1517

Leu Ala Leu He Leu Leu Arg Lys Arg Arg Lys Glu Thr

TTG GCC CTC ATC CTG CTT CGG AAG AGA CGC AAG GAG ACG 1556

Arg Phe Gly Gin Ala Phe Asp Ser Val Met Ala Arg Gly

CGT TTC GGG CAA GCC TTT GAC AGT GTC ATG GCC CGA GGG 1595

Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser Phe Asn

GAG CCA GCT GTA CAC TTC CGG GCA GCC CGA TCT TTC AAT 1634

Arg Glu Arg Pro Glu Arg He Glu Ala Thr Leu Asp Ser

CGA GAA AGG CCT GAA CGC ATT GAG GCC ACA TTG GAT AGC 1673

Leu Gly He Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp

CTG GGC ATC AGC GAT GAA TTG AAG GAA AAG CTG GAG GAT 1712

Val Leu He Pro Glu Gin Gin Phe Thr Leu Gly Arg Met

GTC CTC ATT CCA GAG CAG CAG TTC ACC CTC GGT CGG ATG 1751

Leu Gly Lys Gly Glu Phe Gly Ser Val Arg Glu Ala Gin

TTG GGC AAA GGA GAG TTT GGA TCA GTG CGG GAA GCC CAG 1790

Leu Lys Gin Glu Asp Gly Ser Phe Val Lys Val Ala Val

CTA AAG CAG GAA GAT GGC TCC TTC GTG AAA GTG GCA GTG 1829

Lys Met Leu Lys Ala Asp He He Ala Ser Ser Asp He

AAG ATG CTG AAA GCT GAC ATC ATT GCC TCA AGC GAC ATA 1868

Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe

GAA GAG TTC CTC CGG GAA GCA GCT TGC ATG AAG GAG TTT 1907

Asp His Pro His Val Ala Lys Leu Val Gly Val Ser Leu

GAC CAT CCA CAC GTG GCC AAG CTT GTT GGG GTG AGC CTC 1946

Arg Ser Arg Ala Lys Gly Arg Leu Pro He Pro Met Val

CGG AGC AGG GCT AAA GGT CGT CTC CCC ATT CCC ATG GTC 1985

He Leu Pro Phe Met Lys His Gly Asp Leu His Ala Phe

ATC CTG CCC TTC ATG AAA CAT GGA GAC TTG CAC GCC TTT 2024

Leu Leu Ala Ser Arg He Gly Glu Asn Pro Phe Asn Leu

CTG CTC GCC TCC CGA ATC GGG GAG AAC CCT TTT AAC CTG 2063 Pro Leu Gin Thr Leu Val Arg Phe Met Val Asp He Ala

CCC CTG CAG ACC CTG GTC CGG TTC ATG GTG GAC ATT GCC 2102

Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe He His

TGT GGC ATG GAG TAC CTG AGC TCC CGG AAC TTC ATC CAC 2141

Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Ala Glu Asp

CGA GAC CTA GCA GCT CGG AAT TGC ATG CTG GCC GAG GAC 2180

Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Arg Lys

ATG ACA GTG TGT GTG GCT GAT TTT GGA CTC TCT CGG AAA 2219

He Tyr Ser Gly Asp Tyr Tyr Arg Gin Gly Cys Ala Ser

ATC TAT AGC GGG GAC TAT TAT CGT CAG GGC TGT GCC TCC 2258

Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu Ala

AAA TTG CCC GTC AAG TGG CTG GCC CTG GAG AGC TTG GCT 2297

Asp Asn Leu Tyr Thr Val His Ser Asp Val Trp Ala Phe

GAC AAC TTG TAT ACT GTA CAC AGT GAT GTG TGG GCC TTC 2336

Gly Val Thr Met Trp Glu He Met Thr Arg Gly Gin Thr

GGG GTG ACC ATG TGG GAG ATC ATG ACT CGT GGG CAG ACG 2375

Pro Tyr Ala Gly He Glu Asn Ala Glu He Tyr Asn Tyr

CCA TAT GCT GGC ATT GAA AAT GCT GAG ATT TAC AAC TAC 2414

Leu He Gly Gly Asn Arg Leu Lys Gin Pro Pro Glu Cys

CTC ATC GGC GGG AAC CGC CTG AAG CAG CCT CCG GAG TGC 2453

Met Glu Glu Val Tyr Asp Leu Met Tyr Gin Cys Trp Ser

ATG GAG GAA GTG TAT GAT CTC ATG TAC CAG TGC TGG AGC 2492

Ala Asp Pro Lys Gin Arg Pro Ser Phe Thr Cys Leu Arg

GCC GAC CCC AAG CAG CGC CCA AGC TTC ACG TGT CTG CGA 2531

Met Glu Leu Glu Asn He Leu Gly His Leu Ser Val Leu

ATG GAA CTG GAG AAC ATT CTG GGC CAC CTG TCT GTG CTG 2370

Ser Thr Ser Gin Asp Pro Leu Tyr He Asn He Glu Arg

TCC ACC AGC CAG GAC CCC TTG TAC ATC AAC ATT GAG AGA 2609

Ala Glu Gin Pro Thr Glu Ser Gly Ser Pro Glu Leu His

GCT GAG CAG CCT ACT GAG AGT GGC AGC CCT GAG CTG CAC 2648

Cys Gly Glu Arg Ser Ser Ser Glu Ala Gly Asp Gly Ser

TGT GGA GAG CGA TCC AGC AGC GAG GCA GGG GAC GGC AGT 2687

Gly Val Gly Ala Val Gly Gly He Pro Ser Asp Ser Arg

GGC GTG GGG GCA GTA GGT GGC ATC CCC AGT GAC TCT CGG 2726

Tyr He Phe Ser Pro Gly Gly Leu Ser Glu Ser Pro Gly

TAC ATC TTC AGC CCC GGA GGG CTA TCC GAG TCA CCA GGG 2765

Gin Leu Glu Gin Gin Pro Glu Ser Pro Leu Asn Glu Asn

CAG CTG GAG CAG CAG CCA GAA AGC CCC CTC AAT GAG AAC 2804

Gin Arg Leu Leu Leu Leu Gin Gin Gly Leu Leu Pro His

CAG AGG CTG TTG TTG CTG CAG CAA GGG CTA CTG CCT CAC 2843

Ser Ser Cys

AGT AGC TGT 2852

*

TAACCCTCAGGCAGAGGAAAGTTGGGGCCCCTGGCTCTGCTGACCACTGTGCTGCCTGAC 2912 TAGGCCCAGTCTGATCACAGCCCAGGCAGCAAGGTATGGAGGCTCCTGTGGTAGCCCTCC 2972 CAAGCTGTGCTGGCGCCTGGACGGACCAAATTGCCCAATCCCAGTTCTTCCTGCAGCCGC 3032 TCTGGCCAGCCTGGCATCAGTTCAGGCCTTGGCTTAGAGGAGGTGAGCCAGAGCTGGTTG 3092 CCTGAATGCAGGCAGCTGGCAGGAGGGGAGGGTGGCTATGTTTCCATGGGTACCATGGGT 3152

GTGGATGGCAGTAAGGGAGGGTAGCAACAGCCCTGTGGGCCCCTACCCTCCTGGCTGAGC 3212

TGCTCCTACTTTAGTGCATGCTTGGAGCCGCCTGCAGCCTGGAACTCAGCACTGCCCACC 3272

ACACTTGGGCCGAAATGCCAGGTTTGCCCCTCTTAAGTCACAAAGAGATGTCCATGTATT 3332

GTTCCCTTTTAGGTGATGATTAGGAAGGGATTGGCACACTTGGGTCCCTAAGCCCTATGG 3392

CAGiiAAATGGTGGGATATTCTCAGGTCTGAATCCTCATCATCTTCCTGATTCCCCACCCT 3452

GCAAAGGCCTGGAACTGGCTGTGGGGCTCTGAGGCATGCTGAAGGACAAAAGATTACAGA 3512

GATCCGACTTCAAAAGGCAGGGTCTGAGTCTGGCAGGTGGAGAGGTGCTAAGGGGCTGGC 3572

CCAGGAGTCAGGCATTTCAGGACCCCTCCAAGCTTCTACAGTCTGTCTGAGCATGCTACC 3632

AAGCCCCCAGATACCCCAAAACTAACAGAGGCAGTTTTGTCTGAGCCCAGCCCTCCCACA 3692

TGATGACCCTTAGGTCTACCCTCCTCTCTAAATGGACATCCTCGTTTGTCCCAAGTCTCC 3752

AGAGAGACTACTGATGGCTGATGTGGGTAAGAAAAGTTCCAGGAACCAGGGCTGGGGTGG 3812

AACCAGGGCTGGGGTCGAGGCAGGCTCTTGGGCAGGCTCTTGCTGTTAGGAACATTTCTA 3872

AGCTATTAAGTTGCTGTTTCAAAACAAATAAAATTGAAACATAAAGA. 3919

(9) INFORMATION FOR SEQUENCE ID NO. 8:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2550 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 8:

Ala Gly GCA GGC 6

Leu Lys Leu Met Gly Ala Pro Val Lys Met Thr Val Ser CTG AAG CTC ATG GGC GCC CCA GTG AAG ATG ACC GTG TCT 45

Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val Glu Gly CAG GGG CAG CCA GTG AAG CTC AAC TGC AGC GTG GAG GGG 84

Met Glu Asp Pro Asp He His Trp Met Lys Asp Gly Thr ATG GAG GAC CCT GAC ATC CAC TGG ATG AAG GAT GGC ACC 123

Val Val Gin Asn Ala Ser Gin Val Ser He Ser He Ser GTG GTC CAG AAT GCA AGC CAG GTG TCC ATC TCC ATC AGC 162

Glu His Ser Trp He Gly Leu Leu Ser Leu Lys Ser Val GAG CAC AGC TGG ATT GGC TTA CTC AGC CTA AAG TCA GTG 201

Glu Arg Ser Asp Ala Gly Leu Tyr Trp Cys Gin Val Lys GAG CGG TCT GAT GCT GGC CTG TAC TGG TGC CAG GTG AAG 240

Asp Gly Glu Glu Thr Lys He Ser Gin Ser Val Trp Leu GAT GGG GAG GAA ACC AAG ATC TCT CAG TCA GTA TGG CTC 279

Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu Pro Lys ACT GTC GAA GGT GTG CCA TTC TTC ACA GTG GAA CCA AAA 318

Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin Leu Ser GAT CTG GCG GTG CCA CCC AAT GCC CCT TTT CAG CTG TCT 357

Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr He Tyr TGT GAG GCT GTG GGT CCT CCA GAA CCC GTA ACC ATT TAC 396

Trp Trp Arg Gly Leu Thr Lys Val Gly Gly Pro Ala Pro TGG TGG AGA GGA CTC ACT AAA GTT GGG GGA CCT GCT CCC 435 Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr Gin Arg

TCT CCC TCT GTT TTA AAT GTG ACA GGA GTG ACC CAG CGC 474

Thr Glu Phe Ser Cys Glu Ala Arg Asn He Lys Gly Leu

ACA GAG TTT TCT TGT GAA GCC CGC AAC ATA AAA GGC CTG 513

Ala Thr Ser Arg Pro Ala He Val Arg Leu Gin Ala Pro

GCC ACT TCC CGA CCA GCC ATT GTT CGC CTT CAA GCA CCG 552

Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr He Ser

CCT GCA GCT CCT TTC AAC ACC ACA GTA ACA ACG ATC TCC 591

Ser Tyr Asn Ala Ser Val Ala Trp Val Pro Gly Ala Asp

AGC TAC AAC GCT AGC GTG GCC TGG GTG CCA GGT GCT GAC 630

Gly Leu Ala Leu Leu His Ser Cys Thr Val Gin Val Ala

GGC CTA GCT CTG CTG CAT TCC TGT ACT GTA CAG GTG GCA 669

His Ala Pro Gly Glu Trp Glu Ala Leu Ala Val Val Val

CAC GCC CCA GGA GAA TGG GAG GCC CTT GCT GTT GTG GTT 708

Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu Ala

CCT GTG CCA CCT TTT ACC TGC CTG CTT CGG AAC TTG GCC 747

Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn

CCT GCC ACC AAC TAC AGC CTT AGG GTG CGC TGT GCC AAT 786

Ala Leu Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe

GCC TTG GGC CCT TCT CCC TAC GGC GAC TGG GTG CCC TTT 825

Gin Thr Lys Gly Leu Ala Pro Ala Arg Ala Pro Gin Asn

CAG ACA AAG GGC CTA GCG CCA GCC AGA GCT CCT CAG AAT 864

Phe His Ala He Arg Thr Asp Ser Gly Leu He Leu Glu

TTC CAT GCC ATT CGT ACC GAC TCA GGC CTT ATC CTG GAA 903

Trp Glu Glu Val He Pro Glu Asp Pro Gly Glu Gly Pro

TGG GAA GAA GTG ATT CCT GAG GAC CCT GGG GAA GGC CCC 942

Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Glu Asn Gly

CTA GGA CCT TAT AAG CTG TCC TGG GTC CAA GAA AAT GGA 981

Thr Gin Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn

ACC CAG GAT GAG CTG ATG GTG GAA GGG ACC AGG GCC AAT 1020

Leu Thr Asp Trp Asp Pro Gin Lys Asp Leu He Leu Arg

CTG ACC GAC TGG GAT CCC CAG AAG GAC CTG ATT TTG CGT 1059

Val Cys Ala Ser Asn Ala He Gly Asp Gly Pro Trp Ser

GTG TGT GCC TCC AAT GCA ATT GGT GAT GGG CCC TGG AGT 1098

Gin Pro Leu Val Val Ser Ser His Asp His Ala Gly Arg

CAG CCA CTG GTG GTG TCT TCT CAT GAC CAT GCA GGG AGG 1137

Gin Gly Pro Pro His Ser Arg Thr Ser Trp Val Pro Val

CAG GGC CCT CCC CAC AGC CGC ACA TCC TGG GTG CCT GTG 1176

Val Leu Gly Val Leu Thr Ala Leu He Thr Ala Ala Ala

GTC CTG GGC GTG CTC ACC GCC CTG ATC ACA GCT GCT GCC 1215

Leu Ala Leu He Leu Leu Arg Lys Arg Arg Lys Glu Thr

TTG GCC CTC ATC CTG CTT CGG AAG AGA CGC AAG GAG ACG 1254

Arg Phe Gly Gin Ala Phe Asp Ser Val Met Ala Arg Gly

CGT TTC GGG CAA GCC TTT GAC AGT GTC ATG GCC CGA GGG 1293

Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser Phe Asn

GAG CCA GCT GTA CAC TTC CGG GCA GCC CGA TCT TTC AAT 1332 Arg Glu Arg Pro Glu Arg He Glu Ala Thr Leu Asp Ser

CGA GAA AGG CCT GAA CGC ATT GAG GCC ACA TTG GAT AGC 1371

Leu Gly He Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp

CTG GGC ATC AGC GAT GAA T^G AAG GAA AAG CTG GAG GAT 1410

Val Leu He Pro Glu Gin Gin Phe Thr Leu Gly Arg Met

GTC CTC ATT CCA GAG CAG CAG TTC ACC CTC GGT CGG ATG 1449

Leu Gly Lys Gly Glu Phe Gly Ser Val Arg Glu Ala Gin

TTG GGC AAA GGA GAG TTT GGA TCA GTG CGG GAA GCC CAG 1488

Leu Lys Gin Glu Asp Gly Ser Phe Val Lys Val Ala Val

CTA AAG CAG GAA GAT GGC TCC TTC GTG AAA GTG GCA GTG 1527

Lys Met Leu Lys Ala Asp He He Ala Ser Ser Asp He

AAG ATG CTG AAA GCT GAC ATC ATT GCC TCA AGC GAC ATA 1566

Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe

GAA GAG TTC CTC CGG GAA GCA GCT TGC ATG AAG GAG TTT 1605

Asp His Pro His Val Ala Lys Leu Val Gly Val Ser Leu

GAC CAT CCA CAC GTG GCC AAG CTT GTT GGG GTG AGC CTC 1644

Arg Ser Arg Ala Lys Gly Arg Leu Pro He Pro Met Val

CGG AGC AGG GCT AAA GGT CGT CTC CCC ATT CCC ATG GTC 1683

He Leu Pro Phe Met Lys His Gly Asp Leu His Ala Phe

ATC CTG CCC TTC ATG AAA CAT GGA GAC TTG CAC GCC TTT 1722

Leu Leu Ala Ser Arg He Gly Glu Asn Pro Phe Asn Leu

CTG CTC GCC TCC CGA ATC GGG GAG AAC CCT TTT AAC CTG 1761

Pro Leu Gin Thr Leu Val Arg Phe Met Val Asp He Ala

CCC CTG CAG ACC CTG GTC CGG TTC ATG GTG GAC ATT GCC 1800

Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe He His

TGT GGC ATG GAG TAC CTG AGC TCC CGG AAC TTC ATC CAC 1839

Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Ala Glu Asp

CGA GAC CTA GCA GCT CGG AAT TGC ATG CTG GCC GAG GAC 1878

Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Arg Lys

ATG ACA GTG TGT GTG GCT GAT TTT GGA CTC TCT CGG AAA 1917

He Tyr Ser Gly Asp Tyr Tyr Arg Gin Gly Cys Ala Ser

ATC TAT AGC GGG GAC TAT TAT CGT CAG GGC TGT GCC TCC 1956

Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu Ala

AAA TTG CCC GTC AAG TGG CTG GCC CTG GAG AGC TTG GCT 1995

Asp Asn Leu Tyr Thr Val His Ser Asp Val Trp Ala Phe

GAC AAC TTG TAT ACT GTA CAC AGT GAT GTG TGG GCC TTC 2034

Gly Val Thr Met Trp Glu He Met Thr Arg Gly Gin Thr

GGG GTG ACC ATG TGG GAG ATC ATG ACT CGT GGG CAG ACG 2073

Pro Tyr Ala Gly He Glu Asn Ala Glu He Tyr Asn Tyr

CCA TAT GCT GGC ATT GAA AAT GCT GAG ATT TAC AAC TAC 2112

Leu He Gly Gly Asn Arg Leu Lys Gin Pro Pro Glu Cys

CTC ATC GGC GGG AAC CGC CTG AAG CAG CCT CCG GAG TGC 2151

Met Glu Glu Val Tyr Asp Leu Met Tyr Gin Cys Trp Ser

ATG GAG GAA GTG TAT GAT CTC ATG TAC CAG TGC TGG AGC 2190

Ala Asp Pro Lys Gin Arg Pro Ser Phe Thr Cys Leu Arg

GCC GAC CCC AAG CAG CGC CCA AGC TTC ACG TGT CTG CGA 2229 Met Glu Leu Glu Asn He Leu Gly His Leu Ser Val Leu

ATG GAA CTG GAG AAC ATT CTG GGC CAC CTG TCT GTG CTG 2268

Ser Thr Ser Gin Asp Pro Leu Tyr He Asn He Glu Arg

TCC ^ACC AGC CAG GAC CCC TTG TAC ATC AAC ATT GAG AGA 2307

Ala Clu Gin Pro Thr Glu Ser Gly Ser Pro Glu Leu His

GCT GAG CAG CCT ACT GAG AGT GGC AGC CCT GAG CTG CAC 2346

Cys Gly Glu Arg Ser Ser Ser Glu Ala Gly Asp Gly Ser

TGT GGA GAG CGA TCC AGC AGC GAG GCA GGG GAC GGC AGT 2385

Gly Val Gly Ala Val Gly Gly He Pro Ser Asp Ser Arg

GGC GTG GGG GCA GTA GGT GGC ATC CCC AGT GAC TCT CGG 2424

Tyr He Phe Ser Pro Gly Gly Leu Ser Glu Ser Pro Gly

TAC ATC TTC AGC CCC GGA GGG CTA TCC GAG TCA CCA GGG 2463

Gin Leu Glu Gin Gin Pro Glu Ser Pro Leu Asn Glu Asn

CAG CTG GAG CAG CAG CCA GAA AGC CCC CTC AAT GAG AAC 2502

Gin Arg Leu Leu Leu Leu Gin Gin Gly Leu Leu Pro His

CAG AGG CTG TTG TTG CTG CAG CAA GGG CTA CTG CCT CAC 2541

Ser Ser Cys

AGT AGC TGT 2550

(10) INFORMATION FOR SEQUENCE ID NO. 9:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4364 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 9:

CATTAGATCTTTACATGAAAGTAAAATTTATAAGATTTCTAGAAAGTCAAAAGATGATAA 60 CTATTTCTTAGGATACTAAAAGCACTCACATTATAGAAAAAAAATCAGTTAACTATACTC 120 CACAAACATTAAAGGCTCCCTATAAAAAAACATTTTTAATAGGCAAGCCACAGAAAGGGC 180 AAATATTAATAGTTTGCAATACATATGTATGAAAAGGAATTGAATCTAGAATATTTAACA 240 AAGCTTTACAACTCAAAAAATACAAAGAAAATATTTTTCTTCCAATTGGCAAATTACTTA 300 AACAGAACCTTCACAAAAGAAGATAAGAATGTTTAATAAACATTTGAAGCCATAATAATG 360 ACATCATTAGCCATGATGGAAATGCAAATTTAAGTACCACTTCACATCCACAAGAAAAAG 420 ATAAAAATAAAAGGACTGAGCTCACCAAACATTGGTGAGGATGTGGTAATACTGAAATTC 480 TTGTACCGTGCTCCTGAGGGTATAACATATTACAGGATTTTTTTGAAAACTAGTGGTTCC 540 TTATAAACTTAATGCCCTGGCAACCTCACACCTATTTACTTAAGAATGAAAGGGCCCCGC 600 CCTCCTCCCTCCTCGCTCGCGGGCCGGGCCCGGCATGGTGCGGCGTCGCCGCCGATGGCG 660 CTGAGGCGGAGC 672

Met Gly Arg Pro Gly Leu Pro Pro Leu Pro Leu Pro Pro

ATG GGG CGG CCG GGG CTC CCG CCG CTG CCG CTG CCG CCG 711

Pro Pro Arg Leu Gly Leu Leu Leu Ala Glu Ser Ala Ala

CCA CCG CGG CTC GGG CTG CTG CTG GCG GAG TCC GCC GCC 750

Ala Gly Leu Lys Leu Met Gly Ala Pro Val Lys Leu Thr

GCA GGT CTG AAG CTC ATG GGA GCC CCG GTG AAG CTG ACA 789

Val Ser Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val

GTG TCT CAG GGG CAG CCG GTG AAG CTC AAC TGC AGT GTG 828 Glu Gly Met Glu Glu Pro Asp He Gin Trp Val Lys Asp

GAG GGG ATG GAG GAG CCT GAC ATC CAG TGG GTG AAG GAT 867

Gly Ala Val Val Gin Asn Leu Asp Gin Leu Tyr He Pro

GGG GCT GTG GTC CAG AAC TTG GAC CAG TTG TAC ATC CCA 906

Val Ser Glu Gin His Trp He Gly Phe Leu Ser Leu Lys

GTC AGC GAG CAG CAC TGG ATC GGC TTC CTC AGC CTG AAG 945

Ser Val Glu Arg Ser Asp Ala Gly Arg Tyr Trp Cys Gin

TCA GTG GAG CGC TCT GAC GCC GGC CGG TAC TGG TGC CAG 984

Val Glu Asp Gly Gly Glu Thr Glu He Ser Gin Pro Val

GTG GAG GAT GGG GGT GAA ACC GAG ATC TCC CAG CCA GTG 1023

Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu

TGG CTC ACG GTA GAA GGT GTG CCA TTT TTC ACA GTG GAG 1062

Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin

CCA AAA GAT CTG GCA GTG CCA CCC AAT GCC CCT TTC CAA 1101

Leu Ser Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr

CTG TCT TGT GAG GCT GTG GGT CCC CCT GAA CCT GTT ACC 1140

He Val Trp Trp Arg Gly Thr Thr Lys He Gly Gly Pro

ATT GTC TGG TGG AGA GGA ACT ACG AAG ATC GGG GGA CCC 1179

Ala Pro Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr

GCT CCC TCT CCA TCT GTT TTA AAT GTA ACA GGG GTG ACC 1218

Gin Ser Thr Met Phe Ser Cys Glu Ala His Asn Leu Lys

CAG AGC ACC ATG TTT TCC TGT GAA GCT CAC AAC CTA AAA 1257

Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His Leu Gin

GGC CTG GCC TCT TCT CGC ACA GCC ACT GTT CAC CTT CAA 1296

Ala Leu Pro Ala Ala Pro Phe Asn He Thr Val Thr Lys

GCA CTG CCT GCA GCC CCC TTC AAC ATC ACC GTG ACA AAG 1335

Leu Ser Ser Ser Asn Ala Ser Val Ala Trp Met Pro Gly

CTT TCC AGC AGC AAC GCT AGT GTG GCC TGG ATG CCA GGT 1374

Ala Asp Gly Arg Ala Leu Leu Gin Ser Cys Thr Val Gin

GCT GAT GGC CGA GCT CTG CTA CAG TCC TGT ACA GTT CAG 1413

Val Thr Gin Ala Pro Gly Gly Trp Glu Val Leu Ala Val

GTG ACA CAG GCC CCA GGA GGC TGG GAA GTC CTG GCT GTT 1452

Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asp

GTG GTC CCT GTG CCC CCC TTT ACC TGC CTG CTC CGG GAC 1491

Leu Val Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys

CTG GTG CCT GCC ACC AAC TAC AGC CTC AGG GTG CGC TGT 1530

Ala Asn Ala Leu Gly Pro Ser Pro Tyr Ala Asp Trp Val

GCC AAT GCC TTG GGG CCC TCT CCC TAT GCT GAC TGG GTG 1569

Pro Phe Gin Thr Lys Gly Leu Ala Pro Ala Ser Ala Pro

CCC TTT CAG ACC AAG GGT CTA GCC CCA GCC AGC GCT CCC 1608

Gin Asn Leu His Ala He Arg Thr Asp Ser Gly Leu He

CAA AAC CTC CAT GCC ATC CGC ACA GAT TCA GGC CTC ATC 1647

Leu Glu Trp Glu Glu Val He Pro Glu Ala Pro Leu Glu

TTG GAG TGG GAA GAA GTG ATC CCC GAG GCC CCT TTG GAA 1686

Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Asp

GGC CCC CTG GGA CCC TAC AAA CTG TCC TGG GTT CAA GAC 1725 Asn Gly Thr Gin Asp Glu Leu Thr Val Glu Gly Thr Arg

AAT GGA ACC CAG GAT GAG CTG ACA GTG GAG GGG ACC AGG 1764

Ala Asn Leu Thr Gly Trp Asp Pro Gin Lys Asp Leu He

GCC AAT TTG ACA GGC TGG GAT CCC CAA AAG GAC CTG ATC 1803

Val Arg Val Cys Val Ser Asn Ala Val Gly Cys Gly Pro

GTA CGT GTG TGC GTC TCC AAT GCA GTT GGC TGT GGA CCC 1842

Trp Ser Gin Pro Leu Val Val Ser Ser His Asp Arg Ala

TGG AGT CAG CCA CTG GTG GTC TCT TCT CAT GAC CGT GCA 1881

Gly Gin Gin Gly Pro Pro His Ser Arg Thr Ser Trp Val

GGC CAG CAG GGC CCT CCT CAC AGC CGC ACA TCC TGG GTA 1920

Pro Val Val Leu Gly Val Leu Thr Ala Leu Val Thr Ala

CCT GTG GTC CTT GGT GTG CTA ACG GCC CTG GTG ACG GCT 1959

Ala Ala Leu Ala Leu He Leu Leu Arg Lys Arg Arg Lys

GCT GCC CTG GCC CTC ATC CTG CTT CGA AAG AGA CGG AAA 1998

Glu Thr Arg Phe Gly Gin Ala Phe Asp Ser Val Met Ala

GAG ACG CGG TTT GGG CAA GCC TTT GAC AGT GTC ATG GCC 2037

Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser

CGG GGA GAG CCA GCC GTT CAC TTC CGG GCA GCC CGG TCC 2076

Phe Asn Arg Glu Arg Pro Glu Arg He Glu Ala Thr Leu

TTC AAT CGA GAA AGG CCC GAG CGC ATC GAG GCC ACA TTG 2115

Asp Ser Leu Gly He Ser Asp Glu Leu Lys Glu Lys Leu

GAC AGC TTG GGC ATC AGC GAT GAA CTA AAG GAA AAA CTG 2154

Glu Asp Val Leu He Pro Glu Gin Gin Phe Thr Leu Gly

GAG GAT GTG CTC ATC CCA GAG CAG CAG TTC ACC CTG GGC 2193

Arg Met Leu Gly Lys Gly Glu Phe Gly Ser Val Arg Glu

CGG ATG TTG GGC AAA GGA GAG TTT GGT TCA GTG CGG GAG 2232

Ala Gin Leu Lys Gin Glu Asp Gly Ser Phe Val Lys Val

GCC CAG CTG AAG CAA GAG GAT GGC TCC TTT GTG AAA GTG 2271

Ala Val Lys Met Leu Lys Ala Asp He He Ala Ser Ser

GCT GTG AAG ATG CTG AAA GCT GAC ATC ATT GCC TCA AGC 2310

Asp He Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys

GAC ATT GAA GAG TTC CTC AGG GAA GCA GCT TGC ATG AAG 2349

Glu Phe Asp His Pro His Val Ala Lys Leu Val Gly Val

GAG TTT GAC CAT CCA CAC GTG GCC AAA CTT GTT GGG GTA 2388

Ser Leu Arg Ser Arg Ala Lys Gly Arg Leu Pro He Pro

AGC CTC CGG AGC AGG GCT AAA GGC CGT CTC CCC ATC CCC 2427

Met Val He Leu Pro Phe Met Lys His Gly Asp Leu His

ATG GTC ATC TTG CCC TTC ATG AAG CAT GGG GAC CTG CAT 2466

Ala Phe Leu Leu Ala Ser Arg He Gly Glu Asn Pro Phe

GCC TTC CTG CTC GCC TCC CGG ATT GGG GAG AAC CCC TTT 2505

Asn Leu Pro Leu Gin Thr Leu He Arg Phe Met Val Asp

AAC CTA CCC CTC CAG ACC CTG ATC CGG TTC ATG GTG GAC 2544

He Ala Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe

ATT GCC TGC GGC ATG GAG TAC CTG AGC TCT CGG AAC TTC 2583

He His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Ala

ATC CAC CGA GAC CTG GCT GCT CGG AAT TGC ATG CTG GCA 2622 Glu Asp Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser

GAG GAC ATG ACA GTG TGT GTG GCT GAC TTC GGA CTC TCC 2661

Arg Lys He Tyr Ser Gly Asp Tyr Tyr Arg Gin Gly Cys

CGG AAG ATC TAC AGT GGG GAC TAC TAT CGT CAA GGC TGT 2700

Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser

GCC TCC AAA CTG CCT GTC AAG TGG CTG GCC CTG GAG AGC 2739

Leu Ala Asp Asn Leu Tyr Thr Val Gin Ser Asp Val Trp

CTG GCC GAC AAC CTG TAT ACT GTG CAG AGT GAC GTG TGG 2778

Ala Phe Gly Val Thr Met Trp Glu He Met Thr Arg Gly

GCG TTC GGG GTG ACC ATG TGG GAG ATC ATG ACA CGT GGG 2817

Gin Thr Pro Tyr Ala Gly He Glu Asn Ala Glu He Tyr

CAG ACG CCA TAT GCT GGC ATC GAA AAC GCT GAG ATT TAC 2856

Asn Tyr Leu He Gly Gly Asn Arg Leu Lys Gin Pro Pro

AAC TAC CTC ATT GGC GGG AAC CGC CTG AAA CAG CCT CCG 2895

Glu Cys Met Glu Asp Val Tyr Asp Leu Met Tyr Gin Cys

GAG TGT ATG GAG GAC GTG TAT GAT CTC ATG TAC CAG TGC 2934

Trp Ser Ala Asp Pro Lys Gin Arg Pro Ser Phe Thr Cys

TGG AGT GCT GAC CCC AAG CAG CGC CCG AGC TTT ACT TGT 2973

Leu Arg Met Glu Leu Glu Asn He Leu Gly Gin Leu Ser

CTG CGA ATG GAA CTG GAG AAC ATC TTG GGC CAG CTG TCT 3012

Val Leu Ser Ala Ser Gin Asp Pro Leu Tyr He Asn He

GTG CTA TCT GCC AGC CAG GAC CCC TTA TAC ATC AAC ATC 3051

Glu Arg Ala Glu Glu Pro Thr Val Gly Gly Ser Leu Glu

GAG AGA GCT GAG GAG CCC ACT GTG GGA GGC AGC CTG GAG 3090

Leu Pro Gly Arg Asp Gin Pro Tyr Ser Gly Ala Gly Asp

CTA CCT GGC AGG GAT CAG CCC TAC AGT GGG GCT GGG GAT 3129

Gly Ser Gly Met Gly Ala Val Gly Gly Thr Pro Ser Asp

GGC AGT GGC ATG GGG GCA GTG GGT GGC ACT CCC AGT GAC 3168

Cys Arg Tyr He Leu Thr Pro Gly Gly Leu Ala Glu Gin

TGT CGG TAC ATA CTC ACC CCC GGA GGG CTG GCT GAG CAG 3207

Pro Gly Gin Ala Glu His Gin Pro Glu Ser Pro Leu Asn

CCA GGG CAG GCA GAG CAC CAG CCA GAG AGT CCC CTC AAT 3246

Glu Thr Gin Arg Leu Leu Leu Leu Gin Gin Gly Leu Leu

GAG ACA CAG AGG CTT TTG CTG CTG CAG CAA GGG CTA CTG 3285

Pro His Ser Ser Cys

CCA CAC AGT AGC TGT 3300

TAGCCCACAGGCAGAGGGCATCGGGGCCATTTGGCCGGCTCTGGTGGCCACTGAGCTGGC 3360

TGACTAAGCCCCGTCTGACCCCAGCCCAGACAGCAAGGTGTGGAGGCTCCTGTGGTAGTC 3420

CTCCCAAGCTGTGCTGGGAAGCCCGGACTGACCAAATCACCCAATCCCAGTTCTTCCTGC 3480

AACCACTCTGTGGCCAGCCTGGCATCAGTTTAGGCCTTGGCTTGATGGAAGTGGGCCAGT 3540

CCTGGTTGTCTGAACCCAGGCAGCTGGCAGGAGTGGGGTGGTTATGTTTCCATGGTTACC 3600

ATGGGTGTGGATGGCAGTGTGGGGAGGGCAGGTCCAGCTCTGTGGGCCCTACCCTCCTGC 3660

TGAGCTGCCCCTGCTGCTTAAGTGCATGCATTGAGCTGCCTCCAGCCTGGTGGCCCAGCT 3720

ATTACCACACTTGGGGTTTAAATATCCAGGTGTGCCCCTCCAAGTCAGAAAGAGATGTCC 3780

TTGTAATATTCCCTTTTAGGTGAGGGTTGGTAAGGGGTTGGTATCTCAGGTCTGAATCTT 3840

CACCATCTTTCTGATTCCGCACCCTGCCTACGCCAGGAGAAGTTGAGGGGAGCATGCTTC 3900

CCTGCAGCTGACCGGGTCACACAAAGGCATGCTGGAGTACCCAGCCTATCAGGTGCCCCT 3960

CTTCCAAAGGCAGCGTGCCGAGCCAGCAAGAGGAAGGGGTGCTGTGAGGCTTGCCCAGGA 4020 GCAAGTGAGGCCGGAGAGGAGTTCAGGAACCCTTCTCCATACCCACAATCTGAGCACGCT 4080 ACCAAATCTCAAAATATCCTAAGACTAACAAAGGCAGCTGTGTCTGAGCCCAACCCTTCT 4140 AAACGGTGACCTTTAGTGCCAACTTCCCCTCTAACTGGACAGCCTCTTCTGTCCCAAGTC 4200 TCCAGAGAGAAATCAGGCCTGATGAGGGGGAATTCCTGGAACCTGGACCCCAGCCTTGGT 4260 GGGGGAGCCTCTGGAATGCATGGGGCGGGTCCTAGCTGTTAGGGACATTTCCAAGCT TT 4320 AGTTGCTGTTTAAAATAGAAATAAAATTGAAGACTAAAGACCTA_n 4364

(11) INFORMATION FOR SEQUENCE ID NO. 10:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2550 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 10:

Ala Gly Leu Lys Leu Met Gly Ala Pro Val Lys Leu Thr

GCA GGT CTG AAG CTC ATG GGA GCC CCG GTG AAG CTG ACA 39

Val Ser Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val

GTG TCT CAG GGG CAG CCG GTG AAG CTC AAC TGC AGT GTG 78

Glu Gly Met Glu Glu Pro Asp He Gin Trp Val Lys Asp

GAG GGG ATG GAG GAG CCT GAC ATC CAG TGG GTG AAG GAT 117

Gly Ala Val Val Gin Asn Leu Asp Gin Leu Tyr He Pro

GGG GCT GTG GTC CAG AAC TTG GAC CAG TTG TAC ATC CCA 156

Val Ser Glu Gin His Trp He Gly Phe Leu Ser Leu Lys

GTC AGC GAG CAG CAC TGG ATC GGC TTC CTC AGC CTG AAG 195

Ser Val Glu Arg Ser Asp Ala Gly Arg Tyr Trp Cys Gin

TCA GTG GAG CGC TCT GAC GCC GGC CGG TAC TGG TGC CAG 234

Val Glu Asp Gly Gly Glu Thr Glu He Ser Gin Pro Val

GTG GAG GAT GGG GGT GAA ACC GAG ATC TCC CAG CCA GTG 273

Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu

TGG CTC ACG GTA GAA GGT GTG CCA TTT TTC ACA GTG GAG 312

Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin

CCA AAA GAT CTG GCA GTG CCA CCC AAT GCC CCT TTC CAA 351

Leu Ser Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr

CTG TCT TGT GAG GCT GTG GGT CCC CCT GAA CCT GTT ACC 390

He Val Trp Trp Arg Gly Thr Thr Lys He Gly Gly Pro

ATT GTC TGG TGG AGA GGA ACT ACG AAG ATC GGG GGA CCC 429

Ala Pro Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr

GCT CCC TCT CCA TCT GTT TTA AAT GTA ACA GGG GTG ACC 468

Gin Ser Thr Met Phe Ser Cys Glu Ala His Asn Leu Lys

CAG AGC ACC ATG TTT TCC TGT GAA GCT CAC AAC CTA AAA 507

Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His Leu Gin

GGC CTG GCC TCT TCT CGC ACA GCC ACT GTT CAC CTT CAA 546 Ala Leu Pro Ala Ala Pro Phe Asn He Thr Val Thr Lys

GCA CTG CCT GCA GCC CCC TTC AAC ATC ACC GTG ACA AAG 585

Leu Ser Ser Ser Asn Ala Ser Val Ala Trp Met Pro Gly

CTT TCC AGC AGC AAC GCT AGT TG GCC TGG ATG CCA GGT 624

Ala Asp Gly Arg Ala Leu Leu Gin Ser Cys Thr Val Gin

GCT GAT GGC CGA GCT CTG CTA CAG TCC TGT ACA GTT CAG 663

Val Thr Gin Ala Pro Gly Gly Trp Glu Val Leu Ala Val

GTG ACA CAG GCC CCA GGA GGC TGG GAA GTC CTG GCT GTT 702

Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asp

GTG GTC CCT GTG CCC CCC TTT ACC TGC CTG CTC CGG GAC 741

Leu Val Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys

CTG GTG CCT GCC ACC AAC TAC AGC CTC AGG GTG CGC TGT 780

Ala Asn Ala Leu Gly Pro Ser Pro Tyr Ala Asp Trp Val

GCC AAT GCC TTG GGG CCC TCT CCC TAT GCT GAC TGG GTG 819

Pro Phe Gin Thr Lys Gly Leu Ala Pro Ala Ser Ala Pro

CCC TTT CAG ACC AAG GGT CTA GCC CCA GCC AGC GCT CCC 858

Gin Asn Leu His Ala He Arg Thr Asp Ser Gly Leu He

CAA AAC CTC CAT GCC ATC CGC ACA GAT TCA GGC CTC ATC 897

Leu Glu Trp Glu Glu Val He Pro Glu Ala Pro Leu Glu

TTG GAG TGG GAA GAA GTG ATC CCC GAG GCC CCT TTG GAA 936

Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Asp

GGC CCC CTG GGA CCC TAC AAA CTG TCC TGG GTT CAA GAC 975

Asn Gly Thr Gin Asp Glu Leu Thr Val Glu Gly Thr Arg

AAT GGA ACC CAG GAT GAG CTG ACA GTG GAG GGG ACC AGG 1014

Ala Asn Leu Thr Gly Trp Asp Pro Gin Lys Asp Leu He

GCC AAT TTG ACA GGC TGG GAT CCC CAA AAG GAC CTG ATC 1053

Val Arg Val Cys Val Ser Asn Ala Val Gly Cys Gly Pro

GTA CGT GTG TGC GTC TCC AAT GCA GTT GGC TGT GGA CCC 1092

Trp Ser Gin Pro Leu Val Val Ser Ser His Asp Arg Ala

TGG AGT CAG CCA CTG GTG GTC TCT TCT CAT GAC CGT GCA 1131

Gly Gin Gin Gly Pro Pro His Ser Arg Thr Ser Trp Val

GGC CAG CAG GGC CCT CCT CAC AGC CGC ACA TCC TGG GTA 1170

Pro Val Val Leu Gly Val Leu Thr Ala Leu Val Thr Ala

CCT GTG GTC CTT GGT GTG CTA ACG GCC CTG GTG ACG GCT 1209

Ala Ala Leu Ala Leu He Leu Leu Arg Lys Arg Arg Lys

GCT GCC CTG GCC CTC ATC CTG CTT CGA AAG AGA CGG AAA 1248

Glu Thr Arg Phe Gly Gin Ala Phe Asp Ser Val Met Ala

GAG ACG CGG TTT GGG CAA GCC TTT GAC AGT GTC ATG GCC 1287

Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser

CGG GGA GAG CCA GCC GTT CAC TTC CGG GCA GCC CGG TCC 1326

Phe Asn Arg Glu Arg Pro Glu Arg He Glu Ala Thr Leu

TTC AAT CGA GAA AGG CCC GAG CGC ATC GAG GCC ACA TTG 1365

Asp Ser Leu Gly He Ser Asp Glu Leu Lys Glu Lys Leu

GAC AGC TTG GGC ATC AGC GAT GAA CTA AAG GAA AAA CTG 1404

Glu Asp Val Leu He Pro Glu Gin Gin Phe Thr Leu Gly

GAG GAT GTG CTC ATC CCA GAG CAG CAG TTC ACC CTG GGC 1443 Arg Met Leu Gly Lys Gly Glu Phe Gly Ser Val Arg Glu

CGG ATG TTG GGC AAA GGA GAG TTT GGT TCA GTG CGG GAG 1482

Ala Gin Leu Lys Gin Glu Asp Gly Ser Phe Val Lys Val

GCC CAG CTG AAG CAA GAG GAT GGC TCC TTT GTG AAA GTG 1521

Ala Val Lys Met Leu Lys Ala Asp He He Ala Ser Ser

GCT GTG AAG ATG CTG AAA GCT GAC ATC ATT GCC TCA AGC 1560

Asp He Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys

GAC ATT GAA GAG TTC CTC AGG GAA GCA GCT TGC ATG AAG 1599

Glu Phe Asp His Pro His Val Ala Lys Leu Val Gly Val

GAG TTT GAC CAT CCA CAC GTG GCC AAA CTT GTT GGG GTA 1638

Ser Leu Arg Ser Arg Ala Lys Gly Arg Leu Pro He Pro

AGC CTC CGG AGC AGG GCT AAA GGC CGT CTC CCC ATC CCC 1677

Met Val He Leu Pro Phe Met Lys His Gly Asp Leu His

ATG GTC ATC TTG CCC TTC ATG AAG CAT GGG GAC CTG CAT 1716

Ala Phe Leu Leu Ala Ser Arg He Gly Glu Asn Pro Phe

GCC TTC CTG CTC GCC TCC CGG ATT GGG GAG AAC CCC TTT 1755

Asn Leu Pro Leu Gin Thr Leu He Arg Phe Met Val Asp

AAC CTA CCC CTC CAG ACC CTG ATC CGG TTC ATG GTG GAC 1794

He Ala Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe

ATT GCC TGC GGC ATG GAG TAC CTG AGC TCT CGG AAC TTC 1833

He His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Ala

ATC CAC CGA GAC CTG GCT GCT CGG AAT TGC ATG CTG GCA 1872

Glu Asp Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser

GAG GAC ATG ACA GTG TGT GTG GCT GAC TTC GGA CTC TCC 1911

Arg Lys He Tyr Ser Gly Asp Tyr Tyr Arg Gin Gly Cys

CGG AAG ATC TAC AGT GGG GAC TAC TAT CGT CAA GGC TGT 1950

Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser

GCC TCC AAA CTG CCT GTC AAG TGG CTG GCC CTG GAG AGC 1989

Leu Ala Asp Asn Leu Tyr Thr Val Gin Ser Asp Val Trp

CTG GCC GAC AAC CTG TAT ACT GTG CAG AGT GAC GTG TGG 2028

Ala Phe Gly Val Thr Met Trp Glu He Met Thr Arg Gly

GCG TTC GGG GTG ACC ATG TGG GAG ATC ATG ACA CGT GGG 2067

Gin Thr Pro Tyr Ala Gly He Glu Asn Ala Glu He Tyr

CAG ACG CCA TAT GCT GGC ATC GAA AAC GCT GAG ATT TAC 2106

Asn Tyr Leu He Gly Gly Asn Arg Leu Lys Gin Pro Pro

AAC TAC CTC ATT GGC GGG AAC CGC CTG AAA CAG CCT CCG 2145

Glu Cys Met Glu Asp Val Tyr Asp Leu Met Tyr Gin Cys

GAG TGT ATG GAG GAC GTG TAT GAT CTC ATG TAC CAG TGC 2184

Trp Ser Ala Asp Pro Lys Gin Arg Pro Ser Phe Thr Cys

TGG AGT GCT GAC CCC AAG CAG CGC CCG AGC TTT ACT TGT 2223

Leu Arg Met Glu Leu Glu Asn He Leu Gly Gin Leu Ser

CTG CGA ATG GAA CTG GAG AAC ATC TTG GGC CAG CTG TCT 2262

Val Leu Ser Ala Ser Gin Asp Pro Leu Tyr He Asn He

GTG CTA TCT GCC AGC CAG GAC CCC TTA TAC ATC AAC ATC 2301

Glu Arg Ala Glu Glu Pro Thr Val Gly Gly Ser Leu Glu

GAG AGA GCT GAG GAG CCC ACT GTG GGA GGC AGC CTG GAG 2340 Leu Pro Gly Arg Asp Gin Pro Tyr Ser Gly Ala Gly Asp

CTA CCT GGC AGG GAT CAG CCC TAC AGT GGG GCT GGG GAT 2379

Gly Ser Gly Met Gly Ala Val Gly Gly Thr Pro Ser Asp

GGC AGT GGC ATG GGG GCA GTG GGT GGC ACT CCC AGT GAC 2418

Cys Arg Tyr He Leu Thr Pro Gly Gly Leu Ala Glu Gin

TGT CGG TAC ATA CTC ACC CCC GGA GGG CTG GCT GAG CAG 2457

Pro Gly Gin Ala Glu His Gin Pro Glu Ser Pro Leu Asn

CCA GGG CAG GCA GAG CAC CAG CCA GAG AGT CCC CTC AAT 2496

Glu Thr Gin Arg Leu Leu Leu Leu Gin Gin Gly Leu Leu

GAG ACA CAG AGG CTT TTG CTG CTG CAG CAA GGG CTA CTG 2535

Pro His Ser Ser Cys

CCA CAC AGT AGC TGT 2550

(12) INFORMATION FOR SEQUENCE ID NO. 11:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1158 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 11:

Ala Gly

GCA GGC 6

Leu Lys Leu Met Gly Ala Pro Val Lys Met Thr Val Ser

CTG AAG CTC ATG GGC GCC CCA GTG AAG ATG ACC GTG TCT 45

Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val Glu Gly

CAG GGG CAG CCA GTG AAG CTC AAC TGC AGC GTG GAG GGG 84

Met Glu Asp Pro Asp He His Trp Met Lys Asp Gly Thr

ATG GAG GAC CCT GAC ATC CAC TGG ATG AAG GAT GGC ACC 123

Val Val Gin Asn Ala Ser Gin Val Ser He Ser He Ser

GTG GTC CAG AAT GCA AGC CAG GTG TCC ATC TCC ATC AGC 162

Glu His Ser Trp He Gly Leu Leu Ser Leu Lys Ser Val

GAG CAC AGC TGG ATT GGC TTA CTC AGC CTA AAG TCA GTG 201

Glu Arg Ser Asp Ala Gly Leu Tyr Trp Cys Gin Val Lys

GAG CGG TCT GAT GCT GGC CTG TAC TGG TGC CAG GTG AAG 240

Asp Gly Glu Glu Thr Lys He Ser Gin Ser Val Trp Leu

GAT GGG GAG GAA ACC AAG ATC TCT CAG TCA GTA TGG CTC 279

Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu Pro Lys

ACT GTC GAA GGT GTG CCA TTC TTC ACA GTG GAA CCA AAA 318

Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin Leu Ser

GAT CTG GCG GTG CCA CCC AAT GCC CCT TTT CAG CTG TCT 357

Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr He Tyr

TGT GAG GCT GTG GGT CCT CCA GAA CCC GTA ACC ATT TAC 396 Trp Trp Arg Gly Leu Thr Lys Val Gly Gly Pro Ala Pro

TGG TGG AGA GGA CTC ACT AAA GTT GGG GGA CCT GCT CCC 435

Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr Gin Arg

TCT CCC TCT GTT TTA AAT GTG AC? GGA GTG ACC CAG CGC 474

Thr Glu Phe Ser Cys Glu Ala Arg Asn He Lys Gly Leu

ACA GAG TTT TCT TGT GAA GCC CGC AAC ATA AAA GGC CTG 513

Ala Thr Ser Arg Pro Ala He Val Arg Leu Gin Ala Pro

GCC ACT TCC CGA CCA GCC ATT GTT CGC CTT CAA GCA CCG 552

Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr He Ser

CCT GCA GCT CCT TTC AAC ACC ACA GTA ACA ACG ATC TCC 591

Ser Tyr Asn Ala Ser Val Ala Trp Val Pro Gly Ala Asp

AGC TAC AAC GCT AGC GTG GCC TGG GTG CCA GGT GCT GAC 630

Gly Leu Ala Leu Leu His Ser Cys Thr Val Gin Val Ala

GGC CTA GCT CTG CTG CAT TCC TGT ACT GTA CAG GTG GCA 669

His Ala Pro Gly Glu Trp Glu Ala Leu Ala Val Val Val

CAC GCC CCA GGA GAA TGG GAG GCC CTT GCT GTT GTG GTT 708

Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu Ala

CCT GTG CCA CCT TTT ACC TGC CTG CTT CGG AAC TTG GCC 747

Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn

CCT GCC ACC AAC TAC AGC CTT AGG GTG CGC TGT GCC AAT 786

Ala Leu Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe

GCC TTG GGC CCT TCT CCC TAC GGC GAC TGG GTG CCC TTT 825

Gin Thr Lys Gly Leu Ala Pro Ala Arg Ala Pro Gin Asn

CAG ACA AAG GGC CTA GCG CCA GCC AGA GCT CCT CAG AAT 864

Phe His Ala He Arg Thr Asp Ser Gly Leu He Leu Glu

TTC CAT GCC ATT CGT ACC GAC TCA GGC CTT ATC CTG GAA 903

Trp Glu Glu Val He Pro Glu Asp Pro Gly Glu Gly Pro

TGG GAA GAA GTG ATT CCT GAG GAC CCT GGG GAA GGC CCC 941

Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Glu Asn Gly

CTA GGA CCT TAT AAG CTG TCC TGG GTC CAA GAA AAT GGA 981

Thr Gin Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn

ACC CAG GAT GAG CTG ATG GTG GAA GGG ACC AGG GCC AAT 1020

Leu Thr Asp Trp Asp Pro Gin Lys Asp Leu He Leu Arg

CTG ACC GAC TGG GAT CCC CAG AAG GAC CTG ATT TTG CGT 1059

Val Cys Ala Ser Asn Ala He Gly Asp Gly Pro Trp Ser

GTG TGT GCC TCC AAT GCA ATT GGT GAT GGG CCC TGG AGT 1098

Gin Pro Leu Val Val Ser Ser His Asp His Ala Gly Arg

CAG CCA CTG GTG GTG TCT TCT CAT GAC CAT GCA GGG AGG 1137

Gin Gly Pro Pro His Ser Arg

CAG GGC CCT CCC CAC AGC CGC 1158

(13) INFORM ATION FOR SEQUENCE ID NO. 12:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1158 BASE PAIRS

(B) TYPE: NUCLEIC ACID (C) STRAΝDEDΝESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 12:

Ala Gly Leu Lys Leu Met Gly Ala Pro Val Lys Leu Thr

GCA GGT CTG AAG CTC ATG GGA GCC CCG GTG AAG CTG ACA 39

Val Ser Gin Gly Gin Pro Val Lys Leu Asn Cys Ser Val

GTG TCT CAG GGG CAG CCG GTG AAG CTC AAC TGC AGT GTG 78

Glu Gly Met Glu Glu Pro Asp He Gin Trp Val Lys Asp

GAG GGG ATG GAG GAG CCT GAC ATC CAG TGG GTG AAG GAT 117

Gly Ala Val Val Gin Asn Leu Asp Gin Leu Tyr He Pro

GGG GCT GTG GTC CAG AAC TTG GAC CAG TTG TAC ATC CCA 156

Val Ser Glu Gin His Trp He Gly Phe Leu Ser Leu Lys

GTC AGC GAG CAG CAC TGG ATC GGC TTC CTC AGC CTG AAG 195

Ser Val Glu Arg Ser Asp Ala Gly Arg Tyr Trp Cys Gin

TCA GTG GAG CGC TCT GAC GCC GGC CGG TAC TGG TGC CAG 234

Val Glu Asp Gly Gly Glu Thr Glu He Ser Gin Pro Val

GTG GAG GAT GGG GGT GAA ACC GAG ATC TCC CAG CCA GTG 273

Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu

TGG CTC ACG GTA GAA GGT GTG CCA TTT TTC ACA GTG GAG 312

Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gin

CCA AAA GAT CTG GCA GTG CCA CCC AAT GCC CCT TTC CAA 351

Leu Ser Cys Glu Ala Val Gly Pro Pro Glu Pro Val Thr

CTG TCT TGT GAG GCT GTG GGT CCC CCT GAA CCT GTT ACC 390

He Val Trp Trp Arg Gly Thr Thr Lys He Gly Gly Pro

ATT GTC TGG TGG AGA GGA ACT ACG AAG ATC GGG GGA CCC 429

Ala Pro Ser Pro Ser Val Leu Asn Val Thr Gly Val Thr

GCT CCC TCT CCA TCT GTT TTA AAT GTA ACA GGG GTG ACC 468

Gin Ser Thr Met Phe Ser Cys Glu Ala His Asn Leu Lys

CAG AGC ACC ATG TTT TCC TGT GAA GCT CAC AAC CTA AAA 507

Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His Leu Gin

GGC CTG GCC TCT TCT CGC ACA GCC ACT GTT CAC CTT CAA 546

Ala Leu Pro Ala Ala Pro Phe Asn He Thr Val Thr Lys

GCA CTG CCT GCA GCC CCC TTC AAC ATC ACC GTG ACA AAG 585

Leu Ser Ser Ser Asn Ala Ser Val Ala Trp Met Pro Gly

CTT TCC AGC AGC AAC GCT AGT GTG GCC TGG ATG CCA GGT 624

Ala Asp Gly Arg Ala Leu Leu Gin Ser Cys Thr Val Gin

GCT GAT GGC CGA GCT CTG CTA CAG TCC TGT ACA GTT CAG 663

Val Thr Gin Ala Pro Gly Gly Trp Glu Val Leu Ala Val

GTG ACA CAG GCC CCA GGA GGC TGG GAA GTC CTG GCT GTT 702

Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asp

GTG GTC CCT GTG CCC CCC TTT ACC TGC CTG CTC CGG GAC 741

Leu Val Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys

CTG GTG CCT GCC ACC AAC TAC AGC CTC AGG GTG CGC TGT 780 Ala Asn Ala Leu Gly Pro Ser Pro Tyr Ala Asp Trp Val

GCC AAT GCC TTG GGG CCC TCT CCC TAT GCT GAC TGG GTG 819

Pro Phe Gin Thr Lys Gly Leu Ala Pro Ala Ser Ala Pro

CCC TTT CAG ACC AAG GGT CTA GCC CCA GCC AGC GCT CCC 858

Gin Asn Leu His Ala He Arg Thr Asp Ser Gly Leu He

CAA AAC CTC CAT GCC ATC CGC ACA GAT TCA GGC CTC ATC 897

Leu Glu Trp Glu Glu Val He Pro Glu Ala Pro Leu Glu

TTG GAG TGG GAA GAA GTG ATC CCC GAG GCC CCT TTG GAA 936

Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gin Asp

GGC CCC CTG GGA CCC TAC AAA CTG TCC TGG GTT CAA GAC 975

Asn Gly Thr Gin Asp Glu Leu Thr Val Glu Gly Thr Arg

AAT GGA ACC CAG GAT GAG CTG ACA GTG GAG GGG ACC AGG 1014

Ala Asn Leu Thr Gly Trp Asp Pro Gin Lys Asp Leu He

GCC AAT TTG ACA GGC TGG GAT CCC CAA AAG GAC CTG ATC 1053

Val Arg Val Cys Val Ser Asn Ala Val Gly Cys Gly Pro

GTA CGT GTG TGC GTC TCC AAT GCA GTT GGC TGT GGA CCC 1092

Trp Ser Gin Pro Leu Val Val Ser Ser His Asp Arg Ala

TGG AGT CAG CCA CTG GTG GTC TCT TCT CAT GAC CGT GCA 1131

Gly Gin Gin Gly Pro Pro His Ser Arg

GGC CAG CAG GGC CCT CCT CAC AGC CGC 1158

Claims

CLAIMS:

1. A mammalian receptor tyrosine kinas_^ which is a developmental tyrosine kinase (Dtk) and which is expressed in multipotential haematopoietic cells, in embryonic stem cells, in brain tissue and in testis, but which is not expressed in mature lineage-restricted haematopoietic cells.

2. A receptor tyrosine kinase according to claim 1 that is murine Dtk having the amino acid sequence of SEQ ID NO 1, or a functional equivalent thereof.

3. A receptor tyrosine kinase according to claim 1 that is mature murine Dtk having the amino acid sequence of SEQ ID NO 2.

4. A receptor tyrosine kinase according to claim 1 that is human Dtk having the amino acid sequence of SEQ ID NO 3, or a functional equivalent thereof.

5. A receptor tyrosine kinase according to claim 1 that is mature human Dtk having the amino acid sequence of SEQ ID NO 4.

6. An extracellular receptor domain of a receptor tyrosine kinase according to claim 1.

7. An extracellular receptor domain which is the extracellular receptor domain of mature murine Dtk as defined in claim 3, or a functional equivalent thereof.

8. An extracellular receptor domain of a receptor tyrosine kinase having the amino acid sequence of SEQ ID NO 5.

9. An extracellular receptor domain which is the extracellular receptor domain of mature human Dtk as defined in claim 5, or a functional equivalent thereof.

10. An extracellular receptor domain of a receptor tyrosine kinase having the amino acid sequence of SEQ ID NO 6.

11. An extracellular receptor domain according to any one of claims 6 to 10 which is bound or attached to a support.

12. A soluble receptor comprising the extracellular receptor domain of a receptor tyrosine kinase according to any one of claims 1 to 5 lacking the transmembrane region and catalytic domain of said receptor tyrosine kinase.

13. A nucleic acid molecule encoding a receptor tyrosine kinase as defined in claim 1.

14. A nucleic acid molecule encoding murine Dtk or a functional equivalent thereof as defined in claim 2.

15. A nucleic acid molecule according to claim 14 which is DΝA.

16. A DΝA molecule according to claim 15 having the nucleotide sequence of SEQ ID NO 7.

17. A nucleic acid molecule encoding mature murine Dtk as defined in claim 3.

18. A nucleic acid molecule according to claim 17 which is DNA.

19. A DNA molecule according to claim 18 having the nucleotide sequence of SEQ ID NO 8.

20. A nucleic acid molecule encoding human Dtk or a functional equivalent thereof as defined in claim 4.

21. A nucleic acid molecule according to claim 20 which is DNA.

22. A DNA molecule according to claim 21 having the nucleotide sequence of SEQ ID NO 9.

23. A nucleic acid molecule encoding mature human Dtk as defined in claim 5.

24. A nucleic acid molecule according to claim 23 which is DNA.

25. A DNA molecule according to claim 24 having the nucleotide sequence of SEQ ID NO 10.

26. A nucleic acid molecule encoding an extracellular receptor domain as defined in claim 6.

27. A nucleic acid molecule encoding the extracellular receptor domain of murine Dtk or a functional equivalent thereof as defined in claim 7.

28. A nucleic acid molecule according to claim 27 which is DNA.

29. A DNA molecule according to claim 28 having the nucleotide sequence of SEQ ID NO 11.

30. A nucleic acid molecule encoding the extracellular receptor domain of human Dtk or a functional equivalent thereof as defined in claim 9.

31. A nucleic acid molecule according to claim 30 which is DNA.

32. A DNA molecule according to claim 31 having the nucleotide sequence of SEQ ID NO 12.

33. A vector including a DNA molecule as defined in claim 13.

34. A vector including a DNA molecule as defined in any one of claims 15, 16, 18 and 19.

35. A vector including a DNA molecule as defined in any one of claims 21, 22, 24 and 25.

36. A vector including a DNA molecule as defined in claim 28 or claim 29.

37. A vector including a DNA molecule as defined in claim 31 or claim 32.

38. A method of producing a receptor tyrosine kinase comprising the steps of:

(a) culturing a host cell which has been transformed or transfected with a vector as claimed in any one of claims 33-35 to express the encoded receptor tyrosine kinase; and

(b) recovering the expressed receptor tyrosine kinase.

39. A method of producing an extracellular receptor domain of a receptor tyrosine kinase comprising the steps of:

(a) culturing a host cell which has been transformed or transfected with a vector as claimed in claim 36 or claim 37 to express the encoded extracellular receptor domain; and

(b) recovering the expressed extracellular receptor domain.

40. A recombinant receptor tyrosine kinase which is the product of a method as defined in claim 38.

41. A recombinant extracellular receptor domain which is the product of a method as defined in claim 39.

42. A ligand that binds to a receptor tyrosine kinase as defined in claim 1.

43. A ligand that binds to a receptor tyrosine kinase as defined in claim 2.

44. A ligand that binds to a receptor tyrosine kinase as defined in claim 3.

45. A ligand that binds to a receptor tyrosine kinase as defined in claim 4.

46. A ligand that binds to a receptor tyrosine kinase as defined in claim 5.

47. A ligand that binds to an extracellular receptor domain of a receptor tyrosine kinase as defined in claim 6.

48. A ligand that binds to an extracellular receptor domain of a receptor tyrosine kinase as defined in claim 7.

49. A ligand that binds to an extracellular receptor domain of a receptor tyrosine kinase as defined in claim 8.

50. A ligand that binds to an extracellular receptor domain of a receptor tyrosine kinase as defined in claim 9.

51. A ligand that binds to an extracellular receptor domain of a receptor tyrosine kinase as claimed in claim 10.

52. A ligand that binds to an extracellular receptor domain of a receptor tyrosine kinase as claimed in claim 11.

53. A ligand that binds to a soluble receptor as defined in claim 12.

54. A ligand that binds to a receptor tyrosine kinase as claimed in claim 40.

55. A ligand that binds to an extracellular receptor domain as claimed in claim 41.

56. A ligand according to any one of claims 42-55 wherein the ligand stimulates the proliferation, differentiation and/or survival of cells which express a receptor tyrosine kinase according to claim 1.

57. A ligand according to any one of claims 42-55 wherein the ligand is antagonistic and at least partially blocks or inhibits the function of a receptor tyrosine kinase according to claim 1 through binding to said receptor.

58. A method of stimulating the proliferation, differentiation and/or survival of a cell expressing a receptor tyrosine kinase according to claim 1 comprising contacting the cell with a ligand according to claim 56.

59. A method according to claim 58 wherein the stimulation occurs in vivo.

60. A method according to claim 58 wherein the stimulation occurs ex vivo.

61. A method of inhibiting the function of a receptor tyrosine kinase according to claim 1 comprising contacting the receptor with a ligand according to claim 57.

62. A method according to claim 61 wherein the inhibition occurs in vivo.

63. A method according to claim 61 wherein the inhibition occurs ex vivo.

64. A method of treating a disease, syndrome or condition caused or mediated by an excess of a ligand as claimed in claim 56 comprising the step of contacting said excess of said ligand with an effective amount of a receptor tyrosine kinase according to any one of claims 1-5 and 40, an extracellular receptor domain according to any one of claims 6-11 and 41 or a soluble receptor according to claim 12.

65. A method of treating a disease, syndrome or condition caused or mediated by an excess of a ligand as defined in claim 57 comprising the step of contacting said excess of said ligand with an effective amount of a receptor tyrosine kinase according to any one of claims 1-5 and 40, an extracellular receptor domain according to any one of claims 6-11 and 41 or a soluble receptor according to claim 12.

66. A method of extracting a ligand as defined in claim 56 or claim 57 from a medium which may contain said ligand comprising the step of contacting said medium with a receptor tyrosine kinase according to any one of claims 1-5 and 40, an extracellular receptor domain according to any one of claims 6-11 and 41 or a soluble receptor according to claim 12.

67. A method of isolating ligand(s) as defined in claim 56 or claim 57 from a medium which may contain said ligand(s), comprising the steps of:

(a) contacting said medium with an effective amount of a receptor tyrosine kinase according to any one of claims 1-5 and 40, an extracellular domain according to any one of claims 6-11 and 41 or a soluble receptor according to claim 12; (b) detecting which ligand(s) bind to said tyrosine kinase receptor, extracellular receptor domain or soluble receptor; and (c) isolating such bound ligand(s).

68. A ligand which is isolated by a method according to claim 67.